fkbp52 and its role in dna damage repair · fkbp52 and its role in dna damage repair ii abstract...

FKBP52 AND ITS ROLE IN DNA DAMAGEREPAIR

Nathan Christian Wallace

Bachelor Biomedical Science (Hons)

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Centre for Learning Innovation

Faculty of Health

Queensland University of Technology

2017

FKBP52 and its role in DNA Damage Repair i

Keywords

ATM, ATR, Cancer, Co-Chaperone DNA, DNA Damage Repair, DNA-PKcs,

FKBP52, hSSB1,Homologous Recombination, HSP90, Lung Cancer, NF-κB, p53.

FKBP52 and its role in DNA Damage Repair ii

Abstract

Damage to genetic material represents a constant threat to genomic stability,

with tens of thousands of DNA lesions being produced per day per cell. If DNA

damage is not repaired correctly, it can be detrimental to the cell, leading to alterations

in the genetic material and disease states such as cancer and neurodegenerative

disorders. To protect the integrity of the DNA, cells have evolved a global signalling

network known as the DNA damage response. This network detects different types of

genotoxic stress to raise a versatile and coordinated response, which includes control

of the cell cycle transitions, transcriptional processes and stimulation of DNA repair

and apoptosis.

Recently, the Richard Lab discovered the human single strand DNA binding

protein 1 (hSSB1) to be a crucial component of the DNA damage signalling pathway.

To increase our understanding of the function of hSSB1 in the DNA damage pathway,

a connectivity screen was performed on hSSB1. The connectivity screen is a

bioinformatic tool used to identify mRNA transcripts that are regulated in a similar

manner as the bait (hSSB1). In this study, I sought to identify proteins that may also

be involved in the DNA damage response (DDR) pathway, utilising hSSB1 as the bait.

One of the transcripts identified from this screen was FKBP52. Based on the

previously published literature, this protein has been linked to numerous cancers and

in the regulation of other proteins involved in genome maintenance, such as p53,

hTERT and NF-κB, making it a highly interesting candidate for further study. This

study aims to determine if FKBP52 plays a role in the DDR and if it interacts or

regulates hSSB1.

FKBP52 and its role in DNA Damage Repair iii

FKBP52 is a member of the immunophilin protein superfamily; it consists of a

PPIase domain (peptidylprolyl cis-trans isomerase), a PPIase like domain a TPR

(tetratricopeptide repeat) domain and a calmodulin binding site. The PPIase domain is

required for the isomerisation of peptidyl-prolyl bonds for protein folding to stop the

degradation of partially folded proteins. The TPR domain binds to Heat shock protein

90 (Hsp90) to form a co-chaperone complex. The formation of the co-chaperone

complex allows for the transport and stabilisation of steroid receptors.

To determine if FKBP52 and hSSB1 interact co-immunoprecipitation studies were

performed as well as in vitro direct interaction assays. These showed that FKBP52 and

hSSB1 might co-localise in a complex in vivo but that is was unlikely to be a direct

interaction, leading to the theory that FKBP52 was potentially acting as a co-

chaperone. Supporting this proposition, was the in vivo observation that when FKBP52

levels were depleted by siRNA the corresponding levels of hSSB1 in the nucleus were

reduced and showed little response to IR.

FKBP52 expression was observed to responded to IR induced DNA damage.

FKBP52 levels were also found to increase in the nucleus in response to IR and was

observed to become chromatin associated. found to be bound to the nucleus, this

suggesting it was involved in the DNA damage response. FKBP52 depletion also led

to altered DNA damage signalling in particular with the phosphorylation of key

regulatory proteins of the DNA damage pathway. It was also found that reduced levels

via siRNA led to reduced survival after IR via clonogenic assays and reduced the

efficiency of HR via the HR assay. FKBP52 levels in the nucleus were shown to be

regulated by ATM and ATR consistent with this FKBP52 was found to be

phosphorylated following IR on a putative ATM/ATR phosphorylation site. It was

FKBP52 and its role in DNA Damage Repair iv

found that phosphorylation at this site lead to altered expression of the FKBP52

protein.

CRISPR was utilised to completely remove FKBP52 from the cell over a

longer period. One clone produced a particularly interesting phenotype where both p53

and FKBP52 were completely depleted (referred to as C4-3). This phenotype was

characterised by cells which had an increased resistance to IR and a higher

proliferation rate. The levels of the p53 regulatory proteins MDM2 and p300 were

analysed in the C4-3 cell line. The levels of both proteins were reduced when compared

to the control cell line, and addition of the protesomal inhibitor MG132 lead to the

restoration of both p53 and MDM2, suggesting that these proteins were degraded via

the proteasome in this cell line.

To investigate the role of FKBP52 in a real cancer setting KM plot data was

analysed as well as cancer cell line panels and a lung cancer TMA. It was observed in

the KM data that high FKBP52 mRNA levels indicated a lower probability of survival

in all lung cancer types. When the data was stratified, there was an observable

difference between squamous cell lung cancer and adenocarcinoma lung cancer, were

low levels lead to poorer survival outcomes in squamous small cell lung cancer. The

opposite was true for adenocarcinoma where high levels of FKBP52 mRNA reduced

the probability of survival. Finally, in the lung cancer TMA, it was observed that high

protein high nuclear FKBP52 protein levels overall lead to a lower chance of survival.

This indicates that FKBP52 needs to be carefully regulated in lung cancer cells and

that it could have a potentially important role.

FKBP52 and its role in DNA Damage Repair v

From the data produced it can be seen that FKBP52 is an interesting protein

which responds has an effect on hSSB1 nuclear localisation, responds to DNA damage

and alters the radio sensitivity of cells. This in combination with evidence that FKBP52

is phosphorylated by ATM or ATR in response to IR indicate that FKBP52 could have

a novel role in the DDR. To fully elucidate the role of FKBP52 plays in DNA damage

pathways, further study needs to be performed.

FKBP52 and its role in DNA Damage Repair vi

Table of Contents

Keywords ........................................................................................................................................ i Abstract .......................................................................................................................................... ii Table of Contents ....................................................................................................................... vi List of Figures .............................................................................................................................. ix List of Tables ........................................................................................................................... xvii List of Abbreviations ........................................................................................................... xviii Acknowledgements .............................................................................................................. xxii

Chapter 1: Literature Review and Project Aims. .................................................... 3 1.1 Non-homologous end joining .......................................................................................... 4 1.2 Homologous recombination ............................................................................................ 7 1.3 hSSB1 .................................................................................................................................... 10 1.4 FKBP52................................................................................................................................. 10 1.5 The FKBP co-chaperone mechanism ......................................................................... 12 1.6 Known roles of FKBP52 ................................................................................................. 14 1.7 FKBP52 regulation of steroid hormone receptors ............................................... 15 1.8 FKBP52 in cancer ............................................................................................................. 16 1.9 Interaction between FKBP52, p53 and CK2 kinase ............................................. 17 1.10 FKBP52 and Tauopathies ........................................................................................... 18 1.11 FKBP52 is required for the RISC/Argo complex stability and for effective RNA interference ..................................................................................................................... 19 1.12 FKBP52 is involved in the transport of hTERT to the nucleus ...................... 20 1.13 FKBP52 and Adeno-Associated Viral Vectors...................................................... 21 1.14 FKBP52 is involved in the regulation and transport of NF-κB to the nucleus ........................................................................................................................................ 22

Chapter 2: Materials and Methods ........................................................................... 27 2.1 Materials .............................................................................................................................. 27 2.1.1 General Reagents .......................................................................................................... 27 2.1.2 Antibodies ....................................................................................................................... 27 2.1.2.1 Primary antibodies................................................................................................... 27 2.1.2.2 Western blot secondary antibodies ................................................................... 28 2.1.2.3 Immunofluorescence secondary antibodies ................................................... 28 2.1.3 Enzymes and Kits .......................................................................................................... 29 2.1.4 Cell culture reagents and cell lines ........................................................................ 29 2.1.5 Buffers .............................................................................................................................. 29 2.1.6 Oligonucleotides ........................................................................................................... 30 2.1.7 Plasmids ........................................................................................................................... 31 2.2 Methods ............................................................................................................................... 33 2.2.1 Cell culture ...................................................................................................................... 33 2.2.2 Site directed mutagenesis ......................................................................................... 33 2.2.3 Transfection of plasmid ............................................................................................. 33 2.2.4 siRNA mediated depletion of FKBP52 ................................................................... 34 2.2.5 Clonogenic survival assays ........................................................................................ 34 2.2.6 Immunofluorescence microscopy .......................................................................... 35 2.2.7 Immunoprecipitation .................................................................................................. 35 2.2.8 Flag precipitation ......................................................................................................... 36 2.2.9 Protein extraction ........................................................................................................ 36 2.2.10 Neutral Comet Assay ................................................................................................. 37

FKBP52 and its role in DNA Damage Repair vii

2.2.11 Polymerase chain reaction (PCR) and agarose gel electrophoresis. ....... 37 2.2.12 Western blot analysis ............................................................................................... 38 2.2.13 Homologous Recombination assay ...................................................................... 38 2.2.14 DNA damage induction ............................................................................................ 39 2.2.15 Proliferation assay .................................................................................................... 39 2.2.16 Annexin PI staining ................................................................................................... 39 2.2.17 Protein purification .................................................................................................. 39 2.2.18 Size exclusion- Multi-angle light scattering (MALS) ...................................... 41 2.2.19 Direct protein interaction assay ........................................................................... 41 2.2.20 qRT-PCR ......................................................................................................................... 41 2.2.21 Cell cycle synchronisation double Thymidine block ..................................... 42 2.2.22 Gene editing utilising CRISPR ................................................................................ 42 2.2.23 MG132 Protease inhibition .................................................................................... 43 2.2.24 Sodium butyrate HDAC inhibitor ......................................................................... 43 2.3 Statistics .............................................................................................................................. 44

Chapter 3: Investigating the interaction between FKBP52 and hSSB1 ....... 45 3.1 Introduction ....................................................................................................................... 45 3.2 Validation of antibodies and siRNA ........................................................................... 45 3.3 hSSB1 and FKBP52 are part of the same complex in cell lysates .................... 49 3.4 FKBP52 and hSSB1 do not interact directly in vitro ............................................ 51 3.5 FKBP52 depletion abrogates hSSB1 nuclear localisation ................................. 53

Chapter 4: FKBP52 and its role in DNA damage response ............................... 55 4.1 Introduction ....................................................................................................................... 55 4.2 The FKBP52 is destabilised in response to IR and localises to the nucleus 55 4.3 FKBP52 levels are highest during the G1 phase of the cell cycle. ................... 63 4.4 FKBP52 depletion leads to up regulation of certain DDR proteins ............... 65 4.5 FKBP52 depletion using siRNA leads to radiosensitivity .................................. 67 4.6 FKBP52 overexpression has no effect on apoptosis ............................................ 71 4.7 FKBP52 depletion does not affect cell proliferation ........................................... 74 4.8 FKBP52 depletion has no effect on comet tail length ......................................... 76 4.9 FKBP52 depletion reduces the efficiency of homologous recombination... 78 4.10 Regulation of FKBP52 by ATM/ATR kinases in response to IR .................... 80 4.11 FKBP52 SQSQ sites are phosphorylated in response to IR ............................. 81 4.12 FKBP52 451 and 453 SQ sites phosphorylation state affects the formation of the 44kD band ..................................................................................................................... 86 4.13 Discussion ........................................................................................................................ 88

Chapter 5: Long term depletion of FKBP52 leads to depletion of p53 ........ 96 5.1 Introduction ....................................................................................................................... 96 5.2 Validation of CRISPR clones ......................................................................................... 96 5.3 FKBP52 CRISPR clones show variation in proliferation rate ........................... 99 5.4 CRISPR clones show a radio-resistant phenotype .............................................. 101 5.5 Long term depletion of FKBP52 leads to decreased p53 protein levels .... 103 5.6 FKBP52 depletion causes an increase in p53 and hSSB1 mRNA ................... 105 5.7 Inhibition of the proteasome via MG132 restores p53 protein levels ........ 105 5.8 FKBP52 complementation to restore p53 phenotype ...................................... 112 5.9 Inducible hyperacetylation restores p53 levels ................................................. 114 5.10 Discussion ...................................................................................................................... 117

Chapter 6: FKBP52: a potential survival indicator in lung cancer.............. 120 6.1 Introduction ..................................................................................................................... 120 6.2 Kaplan-Meier data indicates that FKBP52 up or down regulation can be an indicator of survival in cancer .......................................................................................... 120 6.3 FKBP52 protein expression in a lung cancer panel ........................................... 124

FKBP52 and its role in DNA Damage Repair viii

6.4 FKBP52 protein expression in a breast cancer panel ....................................... 124 6.5 TMA data indicates that over or under expression of FKBP52 could indicate poor prognosis ....................................................................................................................... 127 6.6 Discussion ......................................................................................................................... 133

Chapter 7: Discussion ................................................................................................. 135 7.1 FKBP52 and hSSB1......................................................................................................... 135 7.2 The role of FKBP52 in response to IR-induced DNA damage. ........................ 139 7.3 Is FKBP52 regulated by ATM or ATR phosphorylation? .................................. 142 7.4 FKBP52 and lung cancer .............................................................................................. 145 7.5 Model of FKBP52 function in DNA damage response ........................................ 145

Bibliography .................................................................................................................. 151

Appendices ..................................................................................................................... 160 Appendix A ............................................................................................................................... 160 Appendix B ............................................................................................................................... 171

FKBP52 and its role in DNA Damage Repair ix

List of Figures

Figure 1.1 Non-homologous end joining. The DNA ends act as substrates for the binding of Ku70/80 heterodimer, which localises DNA-PKcs to the ends and promotes their juxtaposition. If no further processing of the ends is required, the additional core components of non-homologous DNA end-joining, XRCC4, DNA ligase IV, and XLF can complete the rejoining reaction. Alternatively, end processing may require the activities of the nuclease Artemis and/or the DNA polymerases TdT, pol λ, and pol μ. Figure adapted from Wyman, C. and R. Kanaar (2006). ............................................................................................................. 6

Figure 1.2 Homologous recombination. The MRN complex is initially recruited to sites of DSBs by hSSB1, allowing for resection of the 5’ strand. ssDNA stretches are rapidly bound by RPA, which, following BRCA2-DNA binding, is removed to allow for RAD51 nucleofilament formation. Homologous recombination is then facilitated by RAD51-mediated strand invasion into a sister chromatid. Adapted from Ashton, N. W., et al. (2013). .................................. 9

Figure 1.3 Domains and structure of FKBP52. FKBP52 contains a PPIase domain, a hinge domain regulated by CK2, a PPIase domain with ATP/GTP binding activity, a tetratricopeptide repeat capable of binding to Hsp90, and a Calmodulin binding domain. ................................................... 11

Figure 1.4 Models for steroid hormone receptor translocation. According to the classic model (dashed lines), the chaperone complex dissociates in the cytoplasm from the steroid receptor (SR) on hormone (H) binding. This transformed receptor passes through the nuclear pore complex (NPC) to reach its nuclear sites of action. The novel model is depicted with continuous lines. On steroid binding, the SR heterocomplex exchanges FKBP51 (brown crescent) for FKBP52 (dashed crescent), which can interact with dynein (black). The chaperone complex serves as a traction chain for the receptor, for which retrotransport occurs on cytoskeletal tracts. The nuclear localisation signal (NL1; pink) protrudes on steroid binding and the whole SR–chaperone complex translocates through the NPC. The heterocomplex interacts with structural proteins of the pore, which are also chaperoned. Receptor transformation is nucleoplasmic and facilitates binding of the steroid-activated receptor to promoter sites. Figure retrieved from Storer et.all, 2011 (Storer et al., 2011). ...................... 13

Figure 1.5 Flow chart of the hSSB1 connectivity screen process. The process involves screening online micro array databases to determine genes that are co-regulated with the gene of interest. This screen then produces a list of genes with a probability score. .......................................... 24

Figure 3.1 Optimisation of reagents. Comparison of FKBP52 antibodies. Cells were treated with siRNA targeting FKBP52 or a scramble control (siCtrl), protein was extracted and ran on a 12-4% SDS page gel and transferred to a nitrocellulose membrane. The first blot a) is the Sigma antibody only in grey scale. The second blot b) is the Abcam antibody only in grey scale. Immunofluorescence staining of FKBP52 c) Immunofluorescence of U2OS cells that have been

FKBP52 and its role in DNA Damage Repair x

treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were fixed using 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus. d) Immunofluorescence of U2OS cells that have been treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were pre-extracted to remove -soluble cellular proteins prior to fixation in 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus. f) And g) Depletion of FKBP52 using esiRNA f) and silencer select siRNA g). western blots of U2OS cells transfected with esiRNA targeting FKBP52 or an esiRNA targeting the green fluorescence protein GFP as a control. Cells were harvested at 48,72 and 96 hr post transfection ............................................................................ 47

Figure 3.2 Co-Immunoprecipitation of FKBP52 and hSSB1 a) Co-IP of hSSB1. Anti-hSSB1 goat was cross linked to dyna beads then incubated with cell lysates to remove hSSB1 from solution and any proteins bound to hSSB1. These beads were removed from the solution washed, and boiled to remove hSSB1 form the beads. Samples run on SDS-Page gel and probed with anti-FKBP52 rabbit (Sigma) and hSSB1 goat. b) Anti-FKBP52 rabbit (sigma) was incubated with dynab eads and cell lysates to remove FKBP52 from solution and any proteins bound to FKBP52. These beads were removed from the solution washed, and boiled to remove hSSB1 form the beads. Samples were run on SDS-Page gels and probed with anti-FKBP52 rabbit (sigma) and hSSB1 goat and anti HSP90 goat (abcam). ............................................................... 50

Figure 3.3 FKBP52 and hSSB1 do not interact directly a) Schematic of FKBP52 purification strategy. Cell lysate was centrifuged at 100.000RPM, and loaded on a SP column. Fraction containing FKBP52 were incubated with Ni-NTA resin and following elution the protein was subjected to a size exclusion chromatography b) Recombinant his-FKBP52 1. 2 μg of recombinant FKBP52 were resolved on non reducing SDS-Page gels, and stained using coomassie blue c) FKBP52 exist in dimers in solution. FKBP52 proteins (~200 µg) were applied to a Superdex 200 column with an in line MALLS detector to determine weight-averaged molecular weight in solution. The elution (continuous line) and light-scattering (▪) are shown. d) 2 μg of MBP-hSSB1 and 2μg of his-FKBP52 were incubated overnight at 4 ˚C in K buffer containing 75 mM KCL, and trapped using amylose resin The beads were washed and treated with SDS to elute the bound proteins. The supernatant (S), wash (W), and SDS elute (E) were analysed by SDS-PAGE and stained by Coomassie blue G250. ............................................................................................................. 52

Figure 3.4 FKBP52 and hSSB1 immunofluorescence and cellular localisation after IR a) Immunofluorescence of cells with endogenous (siCtrl) or depleted FKBP52 (siFKBP52) cells were exposed to 6 Gy IR then pre-extracted and fixed at indicated times. Cells probed with DAPI (blue), anti-FKBP52 (red) and anti-hSSB1 (green) antibodies. b) InCell2200 analysis of cell nucleus immunofluorescence intensity comparing siCtrl and siFKBP52 population. Data is graphed as mean +- standard error od mean from 2 independent experiments. T tests were used to compare siCtrl and siFKBP52. ns = > 0.05,* = P < 0.05, ** = P < 0.01, *** = P < 0.001. ..................................................................... 54

FKBP52 and its role in DNA Damage Repair xi

Figure 4.1 FKBP52 response to IR. a) Cells were exposed to 6 Gy IR then protein lysates harvested and run on SDS page gel and western blotted. Blots were probed with anti-FKBP52), anti-FKBP51 and anti-β-actin antibodies. b) Densitometry of FKBP52 full length and 44 kDa bands after exposure to 6 Gy IR. ............................................................................. 57

Figure 4.2 Subcellular localisation of FKBP52 in response to IR. Immunofluorescence microscopy of U2OS cells that have been irradiated with 6 Gy IR. Cells were pre-extracted to remove soluble cellular proteins then fixed using 4% PFA. Cells were probed with antiFKBP52 and γH2AX antibodies and DAPI was used to stain the nucleus (blue). ............................................................................................... 59

Figure 4.3 Sub-cellular localisation of FKBP52 in response to DNA damage Sub cellular fractionation of U2OS cells following exposure to 6 Gy of IR then harvested at the indicated time points using a sub cellular fractionation kit. The fractions shown are: Cytoplasmic, Membrane bound Soluble nuclear and Chromatin and Micrococcal nuclease resistant chromatin fraction. Fractions were probed with anti-Nucleoloin anti-FKBP52 anti-actin and anti-H3 antibodies. ........................ 62

Figure 4.4 FKBP52 levels during the cell cycle a) western blot from cells treated with thymidine to synchronise cells in the G1/S phase. Cells were released and harvested every 4 hr for 24 hr. b) densitometry of western blot FKBP52 upper band normalised to action levels. c) Flow analysis of cells and percentage of cells in G1, S or G2 phase. ........... 64

Figure 4.5 Effect of FKBP52 depletion on DNA damage proteins a) western blot of U2Os cells that have been treated with siControl or siFKBP52 for 72 hr, exposed to 6 Gy IR and harvested at 1, 2, 4 and 6 hours post IR Cells were lysed and proteins harvested and ran on an SDS page gel then transferred to a western blot. westerns were probed with the above antibodies to determine protein levels. ............................................... 66

Figure 4.6 The effect of FKBP52 levels on cell survival after DNA damage a) Clonogenic assay of esiRNA cells treated with IR b) lysates of cells treated with esiRNA targeting FKBP52. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siCtrl and siFKBP52. ns = > 0.05. data was ns. ........ 68

Figure 4.7 Over expression of FKBP52 and radiosensitivity a) Clonogenic survival assay of cells transfected with PCMV6-FLAG vector or PCMV6-FLAG FKBP52 vector. b) western blot presenting cells transfected with PCMV6-FLAG vector or PCMV6-FLAG FKBP52 vector, blot was probed for actin and FKBP52 c) Untreated well of Clonogenic assay was counted and normalised presented as percentage of surviving colonies. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns. .................................................................................................................. 70

Figure 4.8 The effect of FKBP52 on apoptosis and necrosis in Hela cells Cells were transfected with the siRFKBP52 FLAG plasmid then left for 48 hrs. Cells were then harvested at 48 hr and stained with Annexin and PI before flow cytometry a) Graph representing the flow cytometry results n=2 b) Flow cytometry data showing percentage of cells live, Apoptotic Early Apoptotic and Necrotic in Hela cells. Data is graphed as mean +- standard error of the mean from 3 independent

FKBP52 and its role in DNA Damage Repair xii

experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns. ....................................... 72

Figure 4.9 The effect of FKBP52 on apoptosis and necrosis in U20S cells Cells were transfected with the siRFKBP52 FLAG plasmid then left for 48 hrs. Cells were then harvested at 48 hr and stained with Annexin and PI before flow cytometry a) Graph representing the flow cytometry results n=2 b) Flow cytometry data showing percentage of cells live, Apoptotic Early Apoptotic and Necrotic in U20s cells. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns. ....................................... 73

Figure 4.10 FKBP52 has no effect on cell proliferation. U2OS cells were treated with siControl or siFKBP52. Cells were then seeded and monitored every 24 hr and images were taken to determine cell density. Cell numbers were determined using cell profiler data was then graphed to determine proliferation rate of cells data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare sictrl and siFKBP52. ns = > 0.05. data was ns. .................. 75

Figure 4.11 Effect of FKBP52 on DNA damage a) Cells depleted of FKBP52 were irradiated with 6 Gy IR and processed for comet assays. Representative images of comet tails are shown for each sample analysed. b) FKBP52 levels in comet assay lysates c) Graph of measured tail moments for siCtrl and siFKBP52, at untreated (unt), IR+ where sample were harvested immediately after IR and 4hr post IR. Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare sictrl and siFKBP521. ns = > 0.05. data was ns. ........................................................... 77

Figure 4.12 FKBP52 reduces Homologous recombination efficiency. a) flow cytometry of HR assay. Cells were treated with siFKBP52 to deplete FKBP52 levels, then transfected with the ISCE1 plasmid. Cells were left for 24 hours lifted with trypsin then ran through the flow cytometer to detect GFP signal. Blue dots in the gated area indicate GFP expressing cells. b) Graph of normalised Isce1, siCon, siFKBP52 and sihSSB1 flow cytometry data. Data was normalised to GFP transfection Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare siControl and siFKBP521. ns = > 0.05, * = P < 0.01, *** = P < 0.001. ............................................................................................................. 79

Figure 4.13 ATM and ATR phosporylations effect on FKBP52 a) U2OS cells depleted of ATM using siRNA then irradiated with 6 Gy of IR lysates were run on SDS page gel and transferred to a western blot. The blot was probed using anti-FKBP52 rabbit (Sigma) anti-ATM rabbit (Cell signaling) and anti- B-actin mouse (Abcam). b) U2Os cells depleted of ATR using siRNA then irradiated with 6 Gy of IR lysates were run on SDS page gel and transferred to a western blot. The blot was probed using anti-FKBP52 rabbit (Sigma) anti-ATR rabbit (Cell signaling) and anti- B-actin mouse (Abcam). c) U2Os cells transfected with PCMV6 FLAG-FKBP52 plasmid then exposed to 6 Gy IR cells harvested and lysates treated with magnetic flag beads to collect FLAG-FKBP52. Western blot performed with lysates and blot probed with anti-HSP90 goat (Abcam) SQTQ

FKBP52 and its role in DNA Damage Repair xiii

Phospho-(Ser/Thr) ATM/ATR Substrate Antibody mouse (Cell signaling) and anti-FKBP52 rabbit (Sigma). ................................................. 82

Figure 4.14 Knockdown of ATM and ATR and its effect on FKBP52 nuclear levels a) U2Os cells were treated with siRNA to deplete ATM then exposed to 6 Gy IR. Cells were pre-extracted with David’s IF buffer then fixed with 4% PFA at indicated time points. Cells were probed with anti-FKBP52 rabbit (Sigma) and Dapi nuclear stain. b) Nuclear intensity of FKBP52 measured via incell in sicontrol, siATM and siATR populations. Data is graphed as mean +- standard error of mean from 3 independent experiments. T tests were used to compare siCtrl and siATM or siCtrl and siATR. ns = > 0.05. data was ns. ................. 83

Figure 4.15 U2OS cells treated with either DMSO negative control, ATM, ATR inhibitor or both ,30min prior to 6gy IR. Cells were pre-extracted with David’s IF buffer then fixed with 4% PFA at indicated time points. Cells were probed with anti-FKBP52 rabbit (Sigma) anti-γH2AX mouse (abcam) and Dapi nuclear stain. ........................................... 85

Figure 4.16 the effect of phospho active and phospho null mutants on the double SQ domain in FKBP52 a) Diagram of FKBP52 domains including the 451SQ and 453SQ sites. b) U2OS cells were depleted of FKBP52 using siRNA then transfected with no vector, empty flag vector, PCMV6 FLAG FKBP52 siResistant, PCMV6 FLAG FKBP52 siResistant S451A, PCMV6 FLAG FKBP52 siResistant S451E, PCMV6 FLAG FKBP52 siResistant S453A, PCMV6 FLAG FKBP52 siResistant S453E. samples were harvested and ran on an SDS page gel then transferred to a western blot. Blot was probed with FKBP52 and Flag to determine expression of plasmids. c) U2OS cells were depleted of FKBP52 using siRNA then transfected with no vector, empty flag vector, PCMV6 FLAG FKBP52 siResistant, PCMV6 FLAG FKBP52 siResistant S451A, PCMV6 FLAG FKBP52 siResistant S451E, PCMV6 FLAG FKBP52 siResistant S453A, PCMV6 FLAG FKBP52 siResistant S453E and irradiated at 6 Gy. samples were harvested 4 hr post and untreated controls. Samples then ran on an SDS page gel and transferred to a western blot. Blot was probed for FKBP52 and Flag. ................................... 87

Figure 5.1 Crisper Clones targeting exon 1, 4 and 7 a) Hela CRISPRs with controls exon 1 clones C1-3, C1-4, C1-5, C1-6 exon 4 clones C4-1, C4-2 and exon 7 clone C7-3 b) U2OS cells with controls and exon 1 clones C1-1, C1-2, C1-3 and C4-3 some sample performed in duplicate. Blots were probed with sigma FKBP52 to probe FKBP52 levels. ................ 98

Figure 5.2 Proliferation assay of Crisper cell lines. Cells were plated at 3000 cells per well and allowed to grow for 72 hrs. Cells were imaged every 2hrs and confluency was calculated to determine the proliferation rate of each cell line. a) U2OS Crisper cell lines Con, C1-1, C1-2, C1-4 and C4-3 measured over 72hrs. Confluency was normalised to 2hr control to establish base line. b) Hela CRISPR cell lines Con, C1-4 and C4-2 measured over 72 hrs. Confluency was normalised to 2 hr control to establish base line. c) cross section of U2OS CRISPR data focusing on time it takes to reach 2.0 confluency or doubling time. d) cross section of Hela CRISPR data focusing on time it takes to reach 2.0 confluency or doubling time. Experiments were performed in duplicate (n=2) ....................................................................... 100

FKBP52 and its role in DNA Damage Repair xiv

Figure 5.3 The effect of FKBP52 depletion on cell survival after DNA damage a) Clonogenic assay of CRISPR cells treated with IR b) western blot of Clonogenic assay cells and probed using anitFKBP52, values under FKBP52 represent remaining FKBP52 normalised to control. Data is graphed as mean +- standard error of mean from 3 independent experiments. T tests were used to compare U2Os trol and U2Os CRISPR clones or Hela control and Hela CRISPR clones. ns = > 0.05, *= P = < 0.05. ..................................................................................... 102

Figure 5.4 Effect of Crisper Depletion of FKBP52 on DNA damage proteins: western blot of U2Os con, C4-3. Cells were exposed to 6 Gy IR and harvested at 1,2,4 and 6 hrs post IR. Cells were lysed and proteins harvested and ran on an SDS page gel then transferred to a western blot. Westerns were probed with the above antibodies to determine protein levels. .............................................................................................. 104

Figure 5.5 q-RTPCR of U2OS control and U2OS C4-3 CRISPR cells. Cells were exposed to 6gy IR and harvested 1,2,4 and 6 hours post IR. Cells were lysed and RNA was harvested and q-RTPCR was performed with primer pairs targeting FKBP52, hSSB1 and p53 exon junction 2-3 and 8-9. Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare U2Os Control and C4-3. ns = > 0.05, *= P = < 0.05, ** = P < 0.01, *** = P < 0.001. ........................................................................................................ 107

Figure 5.6 Effect of MG132 on P53 and H3 acetylation. U2OS control and C4-3 CRISPR cells. Cell were exposed to DMSO, Mg132 8hrs. cells were then harvested and lysed in RIPA buffer proteins harvested and ran on an SDS page gel then transferred to a western blot. westerns were probed for p300, MDM2, p53 D07, FKBP52, actin, H3 L56 acetylation and H4. ...................................................................................... 108

Figure 5.7 the effect of FKBP52 depletion on nuclear p300 and HDAC1.U2OS Control and U2OS C4-3 CRISPR were fixed with 4% PFA.Cells were probed for FKBP52 HDAC1, p300 and Dapi to stain the nucleus. a) Images of U2OS control CRIPSR and C4-3. incell analysis of the cells measuring the intensity of fluorescence in the CRISPR cell lines U20s con, C1-1, C1-2,C1-4 C4-3, Hela Con, Hela C1-4 and Hela C4-2. b) FKBP52 intensity c) p300 intensity, d) HDAC1 intensity. ........................................................................................ 109

Figure 5.8 the effect of MG132 on the protein levels of p300 and H3K56Ac in FKBP52 depleted cells. U2OS Control and U2OS C4-3 CRISPR were treated with DMSO (control) or MG132 25um for 16hrs then fixed with 4% PFA.Cells were probed for p300 and H3 Lysine 56 acetylation.a) incell anaylsis of the cells measuring the intensity of fluorescence detecting p300. b) incell analysis of the cells measuring the intensity of fluorescence of H3 Lysine 56 acetylation. ......................... 111

Figure 5.9 Attempted rescue of FKBP52 in the C4-3 cell line. U2OS control and C4-3 CRISPR cells were transfected with Flag-FKBP52. (a cells were transfected and after 24hrs cells were exposed to 6 Gy IR then harvested at 1,2,4,and 6hrs post IR. ............................................................ 113

Figure 5.10 Effect of Sodium butyrate (NaBU) on P53 and H3 acetylation. U2OS control and C4-3 CRISPR cells. Cell were exposed to PBS (con) or NaBU for 8hrs. cells were then harvested and lysed in RIPA buffer proteins harvested and ran on an SDS page gel then transferred to a

FKBP52 and its role in DNA Damage Repair xv

western blot. westerns were probed for p300, MDM2, p53 D07, FKBP52, actin, H3 L56 acetylation and H4. ............................................... 115

Figure 5.11 the effect of MG132 on the protein levels of p300 and H3K56Ac in FKBP52 depleted cells. Control and U2OS C4-3 CRISPR were treated with PBS (control) or NaBU 10 mM for 16hrs then fixed with 4% PFA. Cells were probed for p300 and H3 Lysine 56 acetylation. a) incell analysis of the cells measuring the intensity of fluorescence detecting p300. b) Incell analysis of the cells measuring the intensity of fluorescence of H3 Lysine 56 acetylation. ......................... 116

Figure 6.1 FKBP52 in lung and gastric cancer. Kaplan-Meier plots of FKBP52 expression levels and survival of patients with lung cancer, gastric cancer, squamous small cell lung cancer and adenocarcinoma. Km plots were retrieved from kmplot.com. ....................................................... 122

Figure 6.2 FKBP52 in breast cancer. Kaplan-Meier plots of FKBP52 expression levels and survival of patients with Breast Cancer, Breast cancer, was split into Estrogen positive Estrogen negative Progesterone positive and progesterone negative. Km plots were retrieved from kmplot.com. ................................................................................................. 123

Figure 6.3 Expression of FKBP52 in a lung cancer cell line panel. Western blots of FKBP52 and Hsp90 in a panel of lung cancer derived cell lines: HCC827 adenocarcinoma with an EGFR mutation, H2228 adenocarcinoma non-small lung cancer, H460 large cell lung carcinoma, and SKMES squamous cell carcinoma and normal immortalised epithelial cells HBEC3, HBEC4, HBEC5, β-actin was used as loading control. ............................................................................... 125

Figure 6.4 Expression of FKBP52 in a breast cancer cell line panel. Western blot of FKBP52 and HSP90 in breast cancer derived cell lines consisting or the ER positive cell lines: MCF7, T47D, MDA175, MDA361, MDA486, and the ER negative cell lines: MDA468, BT549, BT20 SUM159. The cell lines PMC42ERT and PMC42LA were also included. Western blot was probed for HSP90α, FKBP52 and β-actin. ............................................................................................................ 126

Figure 6.5 TMA of FKBP52 in lung cancer patient samples nuclear staining blue line indicates tumours with a score lower than 150, and the red line indicates tumours with a score greater than 150 with a min of 0 and a max of 300. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only c) squamous count only. ................................. 128

Figure 6.6 TMA of FKBP52 in lung cancer patient samples cytoplasmic staining. Blue line indicates tumours with a score lower than 150, and the red line indicates tumours with a score greater than 150 with a min of 0 and a max of 300. a) combined adenocarcinoma and Squamous count. b) adenocarcinoma count only c) squamous count only ................... 129

Figure 6.7 TMA of FKBP52 in lung cancer patient samples nuclear staining scores have been separated into 0, 1, 2 and 3. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only c) squamous count only. T tests were used to compare 0, 1, 2 and 3. ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001. ............................................. 131

Figure 6.8 TMA of FKBP52 in lung cancer patient samples cytoplasmic staining scores have been separated into 0, 1, 2 and 3. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only

FKBP52 and its role in DNA Damage Repair xvi

c) squamous count only. T tests were used to compare 0, 1, 2 and 3. ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001. ...................... 132

Figure 7.1 Proposed model for FKBP52 response to IR. DNA damage event occurs which in turn leads to the phosphorylation of FKBP52 at S451 and dephoshorylation at S453. This leads to recruitment of hSSB1 and the HSP90 co-chaperone mechanism which in turn transports hSSB1 to the nucleus. .............................................................................................. 149

FKBP52 and its role in DNA Damage Repair xvii

List of Tables

Table 1: - Table contains oligo nucleotide sequences for Site directed mutanogenesis, q-PCR primers and the sequences of siRNAs used in this thesis. ................................................................................................. 32

FKBP52 and its role in DNA Damage Repair xviii

List of Abbreviations

AAV: Adeno-associated virus

ATM: Ataxia telangiectasia mutated

ATR: Ataxia telangiectasia and Rad3 related

AR: Androgen receptor

BRCA1: Breast cancer type 1 susceptibility protein

BRCA2: Breast cancer type 2 susceptibility protein

CA: Clonogenic assay

Cas9: CRISPR associated protein 9

cDNA: complementary DNA

CK2: Casein kinase 2

CRISPR: Clustered regularly interspaced short palindromic repeats

CO-IP: co-immunoprecipitation

DDR: DNA damage repair or DNA damage response

DNA: Deoxyribonucleic acid

DNA-PKcs: DNA-dependent protein kinase, catalytic subunit

DSB: Double strand break

ER: Oestrogen receptor

FACS: Fluorescence-activated cell sorting

FKBP52 and its role in DNA Damage Repair xix

FKBP52: FK506 binding protein 52

FKBP51: FK506 binding protein 51

GFP: Green fluorescent protein

GR: Glucocorticoid receptor

Gy: Grays

H3: Histone 3

H4: Histone 4

HAT: Histone acetyl transferase

HDAC: Histone deacetylases

HSP90: Heat shock protein 90

hSSB1: Human single-stranded DNA-binding protein 1

IF: Immunofluorescence

IP: immunoprecipitation

IR: Ionising radiation

KD: Knock Down

Ku5: ku55933 (ATM inhibitor)

MDM2: mouse double minute 2 homolog

MRN: Mre11-Rad50-Nbs1complex

NaBU: sodium butyrate

NBS1: Nibrin

FKBP52 and its role in DNA Damage Repair xx

NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells

NHEJ: Non-homologous end joining

PAGE: Polyacrylamide Gel Electrophoresis

p300: E1A binding protein p300

PPiase: peptidylprolyl isomerase

PR: Progesterone receptor

RNA: Ribonucleic acid

siRNA: small interfering RNA

SHR: steroid hormone receptor

TPR: tetratricopeptide repeat

VE8: VE821 (ATR inhibitor)

FKBP52 and its role in DNA Damage Repair xxi

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the best

of my knowledge and belief, the thesis contains no material previously published or

written by another person except where due reference is made.

Signature:

Date: September 2017

QUT Verified Signature

FKBP52 and its role in DNA Damage Repair xxii

Acknowledgements

I would like to thank my principal supervisor Derek Richard for giving me this

opportunity. I would also like to thank my other supervisors Laura Croft, Emma

Bolderson and Sally-Anne Stephenson for their support throughout this project. thank

you as well to the CARP group for all the aid that was given to me throughout my time

here as well as their friendship. I especially would like to thank my fellow students

Nicholas Ashton for his willingness to always help me in and out of the lab when I

needed it and Joshua Burgess for all his assistance inside the lab and with life in

general, my Pakistani pal Ali deserves a special mention for all the sweet hangs in the

lab and the good times we shared. I would like to thank my friends and family for

supporting me throughout this trying and arduous time supporting me and encouraging

me to finish my PhD.

Chapter 1: Literature Review and Project Aims. 3

Chapter 1: Literature Review and Project Aims.


with tens of thousands of DNA lesions being produced per day per cell (Bartek &

Lukas, 2007; Lindahl & Barnes, 2000; Wyman & Kanaar, 2006). If DNA damage is

not repaired correctly, it can be detrimental to the cells, with altered structure and

genetic material occurring, leading to disease states such as cancer and

neurodegenerative disorders (Harper & Elledge, 2007; Kerzendorfer & O'Driscoll,

2009). To safeguard the integrity of DNA, cells have evolved a global signalling

network known as the DNA damage response. This detects different types of genotoxic

stress, to raise a versatile and coordinated response, which includes control of the cell

cycle transitions, transcriptional processes, and stimulation of DNA repair (Harrison

& Haber, 2006; Hoeijmakers, 2001; Jackson & Bartek, 2009). The DNA damage

response acts as a major cellular defence against changes that would cause harm to the

cell. The DNA damage response also serves as a check system that functions to

monitor DNA replication, identifying introduced mismatched nucleotides and

collapsed replication forks. At the molecular level, the DNA damage response (DDR)

is organised into a sophisticated system of interacting pathways, the components of

which can be grouped into three major classes of proteins, that act in unison to translate

the signal of damaged DNA into the required downstream response (Kerzendorfer &

O'Driscoll, 2009). As reviewed by Bartek and Lukas (2007), these consist of (1)

sensors, proteins that recognise abnormally structured DNA and initiate the signalling

response, (2) transducers, factors that relay and amplify the damage signal to (3) the

effector proteins in many downstream pathways (Harrison & Haber, 2006; Jackson &

Bartek, 2009; Kerzendorfer & O'Driscoll, 2009).


DNA can be damaged in numerous ways, including single base or nucleotide

modifications and single strand breaks, to the cytotoxic lesions such as inter-strand

crosslinks and double-strand breaks (Bartek & Lukas, 2007; Lieber, 2008). The DNA

damage response (DDR) appears to be most active in the event of a double strand break

(Bartek & Lukas, 2007). Double strand DNA breaks are caused by several endogenous

and exogenous sources, for example ionising radiation (IR), oxidative stress and

replication of damaged DNA. Also, double strand breaks can be formed intentionally

during planned process, such as meiotic recombination and V(D)J recombination in

developing lymphocytes (Bartek & Lukas, 2007). Incorrectly repaired double strand

breaks produce mutations or chromosomal aberrations such as deletions, chromosome

loss or gene translocations. Double strand break repair comprises of two primary

mechanisms, non-homologous end joining (NHEJ) (Lee & Paull, 2005) and

homologous recombination (HR) (Sonoda, Hochegger, Saberi, Taniguchi, & Takeda,

2006). HR occurs during late S or G2 phase of the cell cycle when sister chromatids

are in proximity (Moore & Haber, 1996). NHEJ is the main pathway for the repair of

double strand breaks due to its ability to function throughout the cell cycle and for the

fact that it does not require a homologous chromosome (Moore & Haber, 1996;

Richard et al., 2008).

1.1 Non-homologous end joining

Non-homologous end joining involves the re-joining of what remains of the two

DNA ends, after a double strand break. Although the majority of these re-joining

events will maintain the genetic code, sometimes these ends are no longer compatible

(Lee & Paull, 2005). The repair event must first be preceded by the processing of the

DNA ends (if required), which can cause nucleotide loss or addition of nucleotides at

the re-joining site. Thus, NHEJ is regarded as an imprecise DNA repair pathway, that


can leave “information scars” at the site of repair in vertebrates (Lee & Paull, 2005).

Even though NHEJ can delete or add nucleotides, it is still advantageous due to its

ability to restore the phosphodiester backbone and the structural integrity of the

chromosome. This prevents the deletion of several hundred genes on entire

chromosomal segments or arms that would be lost if repair did not occur (Lee & Paull,

2005).

After occurrence of the double strand break, the Ataxia telangiectasia mutated

kinase (ATM) phosphorylates histone H2AX at Serine 139 (referred to as γH2AX once

phosphorylated). Following this, one Ku protein heterodimer (Ku70/Ku80) binds

tightly at each DNA double strand break termini. The Ku complex is ring-shaped and

acts as a docking site for the protein kinase, DNA-PKcs (Huang, Gong, Ghosal, &

Chen, 2009; Y. Li et al., 2009). DNA-PKcs is then thought to form a bridge between

the DNA ends. This then triggers its auto phosphorylation, catalysing the connection

of both DNA termini (F. Zhang, Wu, & Yu, 2009). This also prevents the DNA from

being prematurely or erroneously degraded. At this point in the process, the Artemis

nuclease is recruited by DNA-PKcs (if needed for double strand break resection).

Artemis can be phosphorylated either by DNA-PKcs or by the ATM kinase (Xu et al.,

2013). Polymerase μ and/or λ may also be recruited to the breakage site and function

to replace nucleotides. This process leads to the inaccuracy of NHEJ (Lee & Paull,

2005). Finally, the scaffolding protein XRCC4 allows DNA Ligase IV to bind to the

DNA allowing the phosphate backbone to be completed. DNA ligase IV function is

enhanced by XLF/Cernunos factor (F. Zhang et al., 2009).


Figure 1.1 Non-homologous end joining. The DNA ends act as substrates for the binding of Ku70/80 heterodimer, which localises DNA-PKcs to the ends and promotes their juxtaposition. If no further processing of the ends is required, the additional core components of non-homologous DNA end-joining, XRCC4, DNA ligase IV, and XLF can complete the rejoining reaction. Alternatively, end processing may require the activities of the nuclease Artemis and/or the DNA polymerases TdT, pol λ, and pol μ. Figure adapted from Wyman, C. and R. Kanaar (2006).


1.2 Homologous recombination

Unlike non-homologous end joining, homologous recombination is restricted to

S and G2 phases of the cell cycle where sister chromatids are present. It uses these as

templates to repair the broken DNA, enabling relatively error-free repair of the break.

The initial detection and signalling event is a sophisticated procedure that begins

with the recognition of a double strand break by hSSB1 (Richard et al., 2008; Richard,

Cubeddu, et al., 2011a). hSSB1 functions to recruit the MRN complex (MRE11-

RAD50-NBS1) to the site of the break. The MRN complex then recruits the ATM

kinase to the damage site, promoting phosphorylation of histone H2AX at the damage

site, launching the signalling cascade (Lee & Paull, 2005; Richard, Cubeddu, et al.,

2011a; Sonoda et al., 2006). This signalling cascade not only amplifies the damage

signal, but recruit’s proteins to the damage site and triggers cell cycle arrest so that

repair can occur. This cascade involves interaction with Mediator of DNA damage

Checkpoint protein 1 (MDC1), ubiquitination of histones by RNF8 and RNF168, and

also the recruitment of RAP80-Abraxas, BRCA1-BRCC36 (Hodge et al., 2016; Huen

et al., 2007; Maréchal & Zou, 2013; Stucki et al., 2005). This results in the 5’ to 3’

resection of the double strand break, generating two single strands of DNA at the break

site. These single strands are initially bound by hSSB1 and Replication Protein A

(RPA), these two proteins function to protect the new single stranded DNA until

RAD51 is ready to commence homologous recombination (Bekker-Jensen & Mailand,

2010; Chun, Buechelmaier, & Powell, 2013; Richard, Cubeddu, et al., 2011a). RAD51

cannot bind to the RPA bound DNA in the absence of BRCA2. BRCA2 functions to

mediate the release of RPA, allowing loading of RAD51 generating an RAD51


nucleoprotein filament that may then invade the sister chromatin (Jie Liu, Doty,

Gibson, & Heyer, 2010).


Figure 1.2 Homologous recombination. The MRN complex is initially recruited to sites of DSBs by hSSB1, allowing for resection of the 5’ strand. ssDNA stretches are rapidly bound by RPA, which, following BRCA2-DNA binding, is removed to allow for RAD51 nucleofilament formation. Homologous recombination is then facilitated by RAD51-mediated strand invasion into a sister chromatid. Adapted from Ashton, N. W., et al. (2013).


1.3 hSSB1

hSSB1 is a 211-residue polypeptide containing an

oligonucleotide/oligosaccharide-binding (OB) fold domain at the N-terminus. hSSB1

has been shown to be a crucial protein in HR (Richard et al., 2008). After DNA

damage, hSSB1 relocates to the DNA damage site and regulates focus formation of

other DNA damage repair proteins, such as BRCA1 and RAD51. Depletion of hSSB1

from cells leads to increased radio-sensitivity, defective checkpoint activation and

genomic instability (Huang et al., 2009; Y. Li et al., 2009; Richard et al., 2008; F.

Zhang et al., 2009). Recent evidence has shown that hSSB1 has a stabilising effect on

proteins involved in the DNA damage signalling pathway such as p53 and p21 and

prevents the ubiquitin degradation of these proteins (Xu et al., 2013). Both of these

proteins are important in cell cycle arrest and apoptosis.

1.4 FKBP52

FKBP52 is a member of the immunophilin protein superfamily, it consists of a

PPIase domain (peptidylprolyl cis-trans isomerase), a PPIase like domain a TPR

(tetratricopeptide repeat) domain and a calmodulin binding site. The PPIase domain is

required for the isomerisation of peptidyl-prolyl bonds throughout protein folding to

stop the degradation of partially folded proteins. The TPR domain binds to Heat shock

protein 90 (Hsp90) to form a co-chaperone complex. The formation of the co-

chaperone complex allows for the transport and stabilisation of steroid receptors, that

are involved in the positive regulation of glucocorticoid (GR), progesterone (PR) and

androgen receptor (AR) but not the oestrogen or mineralocorticoid receptor (Pratt &

Toft, 1997; Storer, Dickey, Galigniana, Rein, & Cox, 2011).


Figure 1.3 Domains and structure of FKBP52. FKBP52 contains a PPIase domain, a hinge domain regulated by CK2, a PPIase domain with ATP/GTP binding activity, a tetratricopeptide repeat capable of binding to Hsp90, and a Calmodulin binding domain figure adapted from (Storer et al., 2011).


1.5 The FKBP co-chaperone mechanism

The current theory of the steroid receptors’ localisation mechanism involves the

signalling molecules binding to Hsp90. HSP90 is then bound to FKBP52 via its TPR

domain (Figure 1.3), while the PPIase domain binds to the dynein motor protein and

in turn the dynein binds to the cytoskeletal “tracks” and shuttles the Hsp90-FKBP52

complex into the nucleus (Storer et al., 2011). This process occurs relatively quickly

[t0.5 =4-5mins] but if the Hsp90-FKBP52 interaction is disrupted for example with

geldanamycin, a Hsp90 inhibitor, this will lower the rate of translocation by order of

magnitude (t0.5 =40-60min). This causes the rapid drop in translocation but not the

cessation of transport, which is thought to be due to active diffusion still allowing the

steroid receptors into the nucleus after the complex has failed. Importantly, when the

nuclear translocation rate of these receptors was impaired, they became highly

sensitive to proteasomal degradation (Galigniana, Harrell, Housley, Patterson, Fisher,

& Pratt, 2004a; Galigniana, Radanyi, Renoir, Housley, & Pratt, 2001). The same

heterocomplex described for steroid receptors is also responsible for cytoplasmic retro

transport of the proapoptotic factor p53 (Galigniana, Harrell, O'Hagen, Ljungman, &

Pratt, 2004b), which suggests that the Hsp90-based complex may play a general role

in the retro transport of a number of Hsp90-associated factors towards the nuclear

surface.


Figure 1.4 Models for steroid hormone receptor translocation. According to the classic model (dashed lines), the chaperone complex dissociates in the cytoplasm from the steroid receptor (SR) on hormone (H) binding. This transformed receptor passes through the nuclear pore complex (NPC) to reach its nuclear sites of action. The novel model is depicted with continuous lines. On steroid binding, the SR heterocomplex exchanges FKBP51 (brown crescent) for FKBP52 (dashed crescent), which can interact with dynein (black). The chaperone complex serves as a traction chain for the receptor, for which retrotransport occurs on cytoskeletal tracts. The nuclear localisation signal (NL1; pink) protrudes on steroid binding and the whole SR–chaperone complex translocates through the NPC. The heterocomplex interacts with structural proteins of the pore, which are also chaperoned. Receptor transformation is nucleoplasmic and facilitates binding of the steroid-activated receptor to promoter sites. Figure from Storer et.all, 2011 (Storer et al., 2011).


1.6 Known roles of FKBP52

FKBP’s are members of the immunophilin protein super family that are

characterised by the ability of the FK506 inhibitor (an immunosuppressant drug) to

bind to the PPIase domain, this inhibits calcineurin signalling, blocking the signal

transduction of T-lymphocytes, The PPIase domain's main function is to accelerate

isomerisation of peptidylprolyl bonds during protein folding to prevent degradation of

partially folded proteins (Baughman, Wiederrecht, Campbell, Martin, & Bourgeois,

1995; Peattie et al., 1992; Storer et al., 2011). The PPIase domain is also used in the

transport of steroid receptors and other signalling molecules to the nucleus. The other

domain that characterises the FKBP family is the TPR (tetratricopeptide repeat)

domain. The TPR domains primary function is to bind to Hsp90 allowing for the

formation of the co-chaperone complex (Peattie et al., 1992; Silverstein et al., 1999;

Storer et al., 2011). The large steroid receptor co-chaperone complex acts by assisting

the maintenance of receptor stability in the absence of ligand and transporting the

receptors to the nuclear membrane. FKBP52 is a positive regulator of the

glucocorticoid receptor (GR) (Banerjee et al., 2008; Davies, Ning, & Sánchez, 2005;

Galigniana et al., 2001; Silverstein et al., 1999), progesterone receptor (PR) (Banerjee

et al., 2008; Cox et al., 2007; Storer et al., 2011), and androgen receptor (AR) (Cheung-

Flynn et al., 2005; De Leon et al., 2011; Yong et al., 2007), but not the estrogen

receptor (ER) (Kumar, Mark, Ward, Minchin, & Ratajczak, 2001; Ward, Mark,

Ingram, Minchin, & Ratajczak, 1999) or mineralocorticoid receptor (MR) (Galigniana,

Erlejman, Monte, Gomez-Sanchez, & Piwien-Pilipuk, 2010; Storer et al., 2011) . The

protein FKBP51 has an antagonistic effect compared to FKBP52 and is a negative

regulator of Steroid Hormone Receptor (SHR) activity in most reported cases. Both

FKBP51 and FKBP52 compete for binding to SHR complexes resulting in a decrease


of regulation by FKBP52 when FKBP51 is up-regulated (Riggs et al., 2003) with the

hormone binding affinity of AR increased five-fold in the presence of FKBP52 (Riggs

et al., 2007).

1.7 FKBP52 regulation of steroid hormone receptors

The glucocorticoid receptor (GR) is the receptor to which glucocorticoids and

cortisol bind. It is ubiquitously expressed and regulates genes involved in

development, the immune response and metabolism. FKBP52 interacts with the GR

through the co-chaperone mechanism and uses the PPIase domain to interact with the

dynein motor protein (Cox et al., 2007) and has been linked with the swift translocation

of GR (Banerjee et al., 2008; Silverstein et al., 1999).

The androgen receptor (AR) has been a major target of androgen deprivation-

based treatments in the clinic and is a key focus in the treatment of prostate cancer.

FKBPs and AR signalling have long been associated, and FKBP52 has been shown to

be regulated in prostate cancer. Male FKBP52-null mice show infertility and reduced

AR activity indicating that FKBP52 is required for proper function of the AR (H. Chen

et al., 2010; Cheung-Flynn et al., 2005; Yong et al., 2007).

FKBP52 is an estrogen-inducible gene that is up-regulated in breast cancer

(Desmetz et al., 2009; Kumar et al., 2001; Ward et al., 1999). More recently, FKBP52

was shown to be specifically methylated in ER-negative, but not ER-positive, breast

cancer cell lines, suggesting that FKBP52 may be involved in breast cancer

progression and/or tumor genesis (Storer et al., 2011).


1.8 FKBP52 in cancer

FKBP52 expression is linked to cancer survival rates, where a high expression

of FKBP52 leads to a lower survival rate, e.g. in bladder, blood, brain, breast, eye and

lung cancer (PrognoScan Data http://www.abren.net/PrognoScan/). Prognoscan is a

database containing mRNA expression and survival data for a number of human

cancers. It has been demonstrated that in prostate cancer cell lines there is an increased

level of FKBP52 protein present and gene knock out strategies have shown that

FKBP52 is a crucial facilitator of physiological androgen receptor activity (De Leon

et al., 2011). FKBP52 is normally ubiquitously expressed at high levels, and stress

conditions lead to a slight up-regulation in the IM-9 cell line. Currently, there is little

known about the role of FKBP52 in cancer. It is possible that FKBP52 has an important

role in hormone related cancers. This is supported by studies in FKBP52-deficient

mice, which show androgen, progesterone and glucocorticoid insensitive phenotypes.

It is possible that FKBP52 could act as a therapeutic target in several diseases that are

dependent on these hormone signal pathways (as reviewed in Storer et.al 2011). It has

also been shown that inhibition of FKBP52’s regulation of androgen receptor function

prevents androgen dependent gene expression and cell proliferation in prostate cancer

cells (De Leon et al., 2011). Furthermore, up-regulation of FKBP52 was discovered

as a marker of drug resistance in breast carcinomas, linking it to another hormone

dependent cancer (Yang et al., 2012).

FKBP52 has also been shown to change the DNA-binding affinity of the

transcription factor IRF-4 (Mamane, Sharma, Grandvaux, Hernandez, & Hiscott,

2002; Mamane, Sharma, Petropoulos, Lin, & Hiscott, 2000). IRF-4 The Interferon

Regulatory Factor 4 (IRF4) gene encodes a transcription factor important for key

developmental stages of hematopoiesis, with known oncogenic implications in

http://www.abren.net/PrognoScan/)


multiple myeloma, adult leukaemias and lymphomas (Adamaki et al., 2013).

Although the interaction between FKBP52 and IRF-4 does not involve their PPIase

domain and DNA-binding domain directly, FKBP52 inhibits the DNA-binding

activity of IRF-4, whereas the inhibition can be reversed by a PPIase inhibitor

(Mamane et al., 2002). These results suggest that FKBP52 is a co-regulator for IRF-

4’s transcriptional activity, possibly through modification of protein factors that

interact with IRF-4. Given its ubiquitous expression, FKBP52 might be an inducible,

transcriptional co-regulator.

FKBP52 has been shown to have possible phosphorylation sites by ATM or ATR

kinases (Matsuoka et al., 2007) This suggests that FKBP52 may be part of the

signalling pathway for DNA damage and repair. FKBP52’s links to cancer and its

already established role in protein stabilisation, regulation of transcription factors and

transport of proteins to the nucleus, lead to a strong case that FKBP52 could also have

a role in DNA damage repair pathways suggesting that further study needs to be

performed to fully understand its function.

1.9 Interaction between FKBP52, p53 and CK2 kinase

FKBP52 has been shown to interact with proteins with already established roles

in the DNA damage and signalling pathway, such as CK2 (casein kinase 2) and p53.

p53 is a tumour suppressor protein that is transported to the nucleus via microtubule

tracks by cytoplasmic dynein. FKBP52 is involved in linking the p53-hsp90 co-

chaperone complex to the dynein motor protein and transporting p53 to the nucleus

and stabilising the protein (Galigniana, Harrell, O'Hagen, Ljungman, & Pratt, 2004b).

Without this mechanism p53 would remain in the nucleus to be polyubiquitinated by

MDM2, which in turn would lead to its degradation (Brook & Gu, 2006).


CK2 was originally found to be elevated in rapidly proliferating cells including

cancer cells. Evidence has shown that CK2 has increased protein expression in all

cancers examined so far. CK2 plays a role in the control of cell growth and

proliferation and on controlling cell death (Miyata, 2009). CK2 phosphorylates

FKBP52 on an evolutionarily conserved threonine residue that only FKBP52 contains

in the linker loop region at aa 143. The phosphorylation appears to lower Hsp90-

binding activity, showing that CK2 phosphorylation weakens FKBP52 co-chaperone

function in-vitro using purified protein. Inversely, in a reticulocyte system phospho-

mimics and phosphorylation incapable mutations to the CK2-phosphorylation site had

no effect on the binding of FKBP52 to Hsp90 compared to the wild type. Therefore it

is still unclear whether CK2 phosphorylation of FKBP52 alters HSP90 binding

(Miyata, 2009).

1.10 FKBP52 and Tauopathies

Tau is a microtubule-associated protein, which is extensively expressed in the

central nervous system primarily in neurones, where it controls microtubule dynamics,

axonal transport and neurite outgrowth. The incorrect assembly of Tau is the most

common feature of numerous neurodegenerative diseases described as tauopathies.

These tauopathies include Alzheimer's disease, Pick's disease, progressive

supranuclear palsy and frontotemporal dementia and parkinsonism linked to

chromosome 17 (Chambraud et al., 2010). Irregularities in Tau disturbs its function

and are key to the pathogenic process. FKBP52, which is highly expressed in the brain,

will specifically and directly bind to Tau especially if Tau is hyper phosphorylated

(which is an aberrant form of Tau). FKBP52 has been shown to prevent the

accumulation of aberrant Tau (Chambraud et al., 2010). Moreover, it has been seen in

Alzheimer’s disease patients, progressive supranuclear palsy, frontotemporal


dementia, parkinsonism linked to chromosome 17 patients, that FKBP52 protein levels

are low compared to normal control patients (Giustiniani et al., 2012). This could

indicate that lack of FKBP52 to regulate Tau may trigger this disease state. This

suggests another important role for FKBP52 in preventing the accumulation of

misfolded proteins and preventing accumulation of plaques in the brain. Interestingly,

Tau has been implicated in the maintenance of genome stability in neuronal cells with

over-expression of TAU resulting in a decrease in DNA damage (Galas, 2016).

1.11 FKBP52 is required for the RISC/Argo complex stability and for effective

RNA interference

Argonaute proteins and small RNAs together form the RNA-induced silencing

complex (RISC), the pivotal component of RNA interference (RNAi). Hsp90 is

required for the crucial step of loading small RNAs onto Argonaute proteins. Data

shows that Hsp90, FKBP52 and p23 form a stable complex with human Ago2 before

small RNA loading (Pare, LaPointe, & Hobman, 2013). It has also been shown that

FKBP52 is required for efficient RNAi and that knocking down FKBP52 leads to a

decrease in Ago2 and therefore is needed for the whole RNAi process to proceed

(Martinez, Chang, Borrajo, & Gregory, 2013). The importance of FKBP52 in the

RISC pathway shows how this protein appears to be incorporated in many conserved

and integral regulatory mechanisms, and that it can have many diverse roles

throughout the cell.

FKBP52 functions in important regulatory pathways and appears essential for

many cellular processes. These can only strengthen the argument that FKBP52 could

have an important role in DNA damage signalling and repair, possibly through the co-

chaperone mechanism that also appears in many different processes or possibly it


could be through the activity of its PPIase domain, which stabilises and activates many

proteins in the cell.

1.12 FKBP52 is involved in the transport of hTERT to the nucleus

Human telomerase reverse transcriptase (hTERT) is a component of the

telomerase that in combination with the telomerase RNA component (TERC) form a

critical element of the telomerase complex. Without telomerase activity, the telomeres

of the DNA would shorten after each round of cellular division. Shortening of the

telomeres can lead to cellular senescence and chromosome instability (Jafri, Ansari,

Alqahtani, & Shay, 2016). Telomerase can lead to cell immortality and is expressed in

70-90% of immortal cell lines and malignant tissues, while it is suppressed in the

majority of somatic tissue. Lagadari et.al. (2016) investigated the potential role of the

HSP90 co-chaperones FKBP51 and FKBP52 and if they interacted with or regulated

hTERT (Lagadari, Zgajnar, Gallo, & Galigniana, 2016). The study found that both

FKBP51 and FKBP52 co-immunoprecipitate with hTERT. Attempts to inhibit this

using Radicicol (an HSP90 inhibitor), disrupts the heterocomplex and favours the

partial cytoplasmic relocalisation of hTERT. This is comparable to the over expression

of the TPR-domain of FKBP proteins. Oxidative stress appears to cause FKBP51

localisation to the nucleus but not FKBP52. It was also found the hTERT activity was

enhanced by both FKBP51 and FKBP52. The proposed mechanism infers that

FKBP52 is required for the retro transport of hTERT through dynein/dynactin. Once

inside the nucleus FKBP51 replaces FKBP52 and enhances hTERT activity (Lagadari

et al., 2016). The hTERT transport mechanism was further elucidated by Jong et al.,

(2016) showing that FKBP52 is required for the interaction with the dynein-dynactin

motor protein complex, and subsequent transport of hTERT into the nucleus. When

FKBP52 was depleted hTERT nuclear transport could not occur, leading to its


accumulation in the cytoplasm and subsequent ubiquitination-mediated degradation.

This leads to decreased telomerase activity (Jeong, Her, Oh, & Chung, 2016). Overall

the study shows a molecular mechanism where FKBP52 regulates telomerase activity

through promoting dynactin-dependent nuclear transport of hTERT.

1.13 FKBP52 and Adeno-Associated Viral Vectors

Adeno-associated viruses (AAV) are a non-enveloped replication-defective

animal virus of approximately 20 nm in diameter. They belong to Dependovirus, a

genus of the family Parvoviridae. AAV replication requires co-infection of a helper,

for example, a herpesvirus or adenovirus. These parvoviruses are non-pathogenic to

humans and have currently been repurposed as retrovirus and adenovirus-based

vectors in gene transfer therapies (Berns & Giraud, 1996a; 1996b; Muzyczka, 1992).

The life cycle of Adenoviruses involves: viral binding, entry, intracellular trafficking,

uncoating, second strand DNA synthesis and viral genome integration into a host

genome. Second strand synthesis appears to be a rate-limiting step that FKBP52 is

involved in (Qing et al., 2003; Zhong et al., 2007). FKBP52 interacts with the single

stranded D-sequence in the AAV2 inverted terminal repeat. FKBP52 is

phosphorylated at tyrosine residues by epidermal growth factor protein tyrosine kinase

(EGFR). This inhibits the viral second-strand DNA synthesis and leads to inefficient

transgene expression (Qing et al., 2003; Zhong et al., 2007). It has also been reported

that FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine

phosphatase (TC-PTP), and this negatively regulates EGFR signalling. This

dephosphorylation, in turn, leads to efficient viral second-strand DNA synthesis.

Tyrosine-dephosphorylation of FKBP52 in TC-PTP-transgenic mice, and deletion of


FKBP52 in fkbp52-/- knockout mice also leads to efficient AAV2 transduction of

murine hepatocytes in vivo (Zhong et al., 2007).

AAV replication studies in vitro have identified DNA polymerase (delta) to

catalyse recombinant adeno associated viral vectors (rAAV) and fills single stranded

DNA gaps created by nucleotide excision repair (NER). The MRN complex

(described in section 1.2) has been shown to be activated upon rAAV infection and

bind to the AAV-ITR (adeno associated viral vectors inverted terminal repeats), which

inhibits wtAVV2 replication and rAAV transduction. ATM is another protein that

potentially could be involved in this process of single to double strand DNA synthesis

as transduction efficiency with ss rAAV is significantly enhanced in ATM deficient

cells in vitro (Schwartz et al., 2007). Given the role of FKBP52 in the conversion of

single stranded DNA (ssDNA) to double stranded DNA in the second strand synthesis

process of viral integration, it could be postulated that FKBP52 may also play a role

in the DDR pathway in a non-viral context.

1.14 FKBP52 is involved in the regulation and transport of NF-κB to the nucleus

Nuclear factor kappa light chain enhancer of activated B cells (NF-κB)

encompasses a family of closely related transcription factors which regulate the

expression of a large number of genes in relation to several processes for example:

inflammatory responses, cell growth, immune responses, cell development, synaptic

plasticity, memory, cancer processes (Oeckinghaus & Ghosh, 2009). This group of

transcription factors is part of a swift acting set of cell factors which are activated by

a sizeable assortment of signals and stresses such as; reactive oxygen species, ionising

radiation, UV light, cell injuries and so on. NF-κB consists of a family of structurally

related homologues that incorporate p50 (NF-κB1), p52 (NF-κB2), p65 ( Rel A), Rel


B, and c-Rel (Oeckinghaus & Ghosh, 2009). All homologues contain a DNA binding

and dimerization domain that is conserved throughout the family. These proteins are

theoretically able to form 15 potential dimers, but to date, not all have been discovered,

the dominant dimer in all cell types is p50.p65/RelA (Oeckinghaus & Ghosh, 2009).

With NF-κB requiring nuclear transport to become activated, Galigniana et al., (2014)

recently investigated FKBP51, FKBP52 and NF-κB regulation. It was found that

FKBP52 and FKBP51 both affect the nuclear translocation of RelA/p65 complex and

also regulate the transcriptional activity of NF-κB. When FKBP51 binds to NF-κB,

the transcription activity of NF-κB is inhibited and the nuclear translocation of

p50.p65/RelA complex is hindered. FKBP52 appears to have an antagonistic effect

and up regulates transport of NF-κB to the nucleus and consequently up-regulates

transcription of target genes (Erlejman et al., 2014). By creating inactive TPR domain-

mutants of FKBP52, it was found that these effects were independent of the HSP90

co-chaperone mechanism that the FKBP immunophilins are known to be involved in.

This presents a novel pathway of immunophilin transport (Lagadari, De Leo, Camisay,

Galigniana, & Erlejman, 2015). This may implicate a role of FKBP52 and FKBP51 in

a cancer through the NF-κB pathway.

1.15 Aims of this study

FKBP52 was identified using a connectivity map screen with hSSB1 as the bait.

Connectivity maps are bioinformatics-based screens looking at genes that are co- or

inversely regulated with the bait gene.


On identification of FKBP52, a literature review was performed to determine

any putative already established links between FKBP52 and DNA damage repair

pathways. Targeting the DNA damage response pathways in cancer cells is recognised

as a major therapeutic intervention in a number of cancers. Indeed, most of the first

line chemotherapeutics function by causing genomic damage to the cancer cells. In

combination with being identified as potentially upregulated gene with hSSB1,

FKBP52 has already been shown to have a role in hormonal cancers and the transport

of proteins known to be part of the DNA damage response such as p53, NF-κB and

recently hTERT (Galigniana et. al, 2014. Chung et. al, 2016) . It could be postulated

that a potential role of FKBP52 may be in the transport mechanism of target proteins

in response to stress to allow these proteins into the nucleus to activate the DNA

damage response pathway. Potentially FKBP52 may have a similar role of transporting

hSSB1 or other DNA damage response proteins into the nucleus following DNA

damage. Therefore, it is hypothesised that FKBP52 may play a role in the DNA

damage response pathway. FKBP52 may interact with hSSB1, either through the co-

Figure 1.5 Flow chart of the hSSB1 connectivity screen process. The process involves screening online microarray databases to determine genes that are co-regulated with the gene of interest. This screen then produces a list of genes with a probability score


chaperone mechanism or independently, and transport hSSB1 to the nucleus in

response to DNA damage. More specifically, the aims of the project are:

1. To investigate the interaction between hSSB1 and FKBP52 in vivo and in vitro.

2. To investigate the role of FKBP52 in response to IR-induced DNA damage.

3. To investigate the potential role of FKBP52 in cancer.

.

Chapter 2: Materials and Methods 27

Chapter 2: Materials and Methods

2.1 Materials

2.1.1 General Reagents

Optimem, Lipofectamine 2000 and Lipofectamine RNAiMAX and the Bolt tm 4-

12% Bis-Tris Plus SDS-Page gels, 10 and 15 wells, were all purchased from

Invitrogen. Phosphatase inhibitor and Methylene blue were purchased from Sigma-

Aldrich. PIC (100X) was purchased from Cell Signalling Technologies KU-57788 was

purchased from Jomar Life Research, all other chemicals were purchased from Sigma-

Aldrich unless otherwise stated.

2.1.2 Antibodies

2.1.2.1 Primary antibodies

The following primary antibodies were used in this study.

Anti-FKBP4 antibody produced in rabbit, Anti-FKBP5 antibody produced in

rabbit, Anti –p53 mouse monoclonal DO-7 and Monoclonal Anti-Flag M2 mouse were

purchased from Sigma-Aldrich.

Mouse monoclonal [9F3] to gamma H2A.X (phosphor S139) and anti-Banf1

mouse monoclonal were purchased from Abcam.

Anti-P-Brca-1 S1524 produced in rabbit, anti-P-ATR (pSer428), anti-P-ATM

s1981 produced in rabbit, anti-NF-ΚB p65 produced in rabbit, anti-P-p53 S15 rabbit,


anti-P-chk2 T68 rabbit and anti-SQTQ Phospho-(Ser/Thr) ATM/ATR Substrate

Antibody produced in mouse were purchased from Cell signalling.

Anti-ATM produced in rabbit was purchased from Cal Biochem.

Anti-HSP90α produced in goat, anti-BRCA1 produced in rabbit and anti-

RAD51 produced in rabbit were purchased from Santa Cruz.

Anti β-Actin produced in mouse was purchased from BD Biosciences. Anti-

hSSB1 produced in sheep was purified from anti-serum described previously (D.J.

Richard et al., 2008) in house.

2.1.2.2 Western blot secondary antibodies

Anti-Rabbit IgG antibody, IR Dye 680RD conjugated produced in donkey, Anti-

Mouse IgG antibody, IR Dye 680RD conjugated produced in donkey, Anti-Goat IgG

antibody, IR Dye 680RD conjugated produced in donkey, Anti-Rabbit IgG antibody,

IR Dye 800RD conjugated produced in donkey, Anti-Mouse IgG antibody, IR Dye

800RD conjugated produced in donkey, anti-Goat IgG antibody, IR Dye 800RD

conjugated produced in donkey were purchased from Licor Bio-sciences.

2.1.2.3 Immunofluorescence secondary antibodies

Alexa Fluor 594 donkey anti-rabbit IgG (H+L), Alexa Fluor 594 donkey anti-

mouse IgG (H+L), Alexa Fluor 594 donkey anti-sheep IgG (H+L), Alexa Fluor 488

donkey anti-rabbit IgG (H+L), Alexa Fluor 488 donkey anti-mouse IgG (H+L) and

Alexa Fluor 488 donkey anti-sheep IgG (H+L) were purchased from ThermoFisher

Scientific.


2.1.3 Enzymes and Kits

Bicinchoninic Acid Protein Determination Kit was purchased from Sigma-Aldrich.

The Sub Cellular Fractionation Kit was purchased from Thermo-Fisher Scientific. The

Isolate II plasmid Mini Kit 250 Preps kit was purchased from Bioline. The Annexin

V-FITC apoptosis detection kit was purchased from United Bioresearch.

2.1.4 Cell culture reagents and cell lines

Human osteosarcoma (U2OS) cells and cervical carcinoma (HeLa) cells were

purchased from ATCC. Roswell Park Memorial Institute medium (RPMI) and Foetal

Bovine Serum were purchased from (Sigma-Aldrich). Trypsin 0.5% EDTA, was

purchased from Invitrogen.

2.1.5 Buffers

PBS: - 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 .

10X ATM transfer buffer: - Tris 60g, Glycine 30g, 1 litre water. (1x add 200ml

ethanol, 100ml buffer 700ml water)

MES running buffer: - 50mM MES, 50 mM Tris Base, 0.1% SDS, 1mM EDTA, pH

7.3.


MOPS running buffer: - MOPs 50mM, 50mM Tris Base, 0.1% SDS, 1mM EDTYA,

pH 7.7.

Phospho lysis buffer: - Tris HCL 50mM, pH 7.4, EDTA 1mM, NaCl 150mM, NP40

1% NaF 5mM, Na deoxycholate 0.25%.

IP and GST pull down buffer: - 20mM HEPES pH8, 150mM KCL, 5% glycerol,

10mM MgCl2, 0.5mM EDTA, .2% NP40.

Comet lysis buffer: - 2.5M NaCl, 100mM EDTA, 10mM Tris (pH10), 1% Triton X-

100.

Immunofluorescence buffer: - 20mM HEPES pH8, 20mM NaCl, 5mM MgCl2, 0.5%

IGEPAL.

TBE: - TRIS base 1M, Boric acid 1M, EDTA 0.02M.

4X SDS protein sample buffer: - 40% Glycerol, 240mM TRIS-HCl pH 6.8, 8% SDS,

0.04% bromophenol blue, 5% beta- mercaptoethanol.

LB buffer: - 10 g tryptone, 5 g yeast extract, 5 g NaCl, 1 Litre water.

2.1.6 Oligonucleotides

All nucleotides used for Site directed mutanagenesis (SDM) were purchased from

Sigma-Aldrich, there sequences can be found in table 1. siFKBP4 esiRNA was


purchased from Sigma- Aldrich. siFKBP52 silencer select siATM stealth and siATR

stealth were all purchased from Invitrogen.

2.1.7 Plasmids

The mammalian expression vectors containing the FLAG-FKBP4 (pcDNA3.1+-DYK)

and FLAG-FKBP4-siR containing the siRNA resistant sequence were purchased from

Origene.


Table 1: - Table contains oligo nucleotide sequences for Site directed mutagenesis, q-PCR primers and

the sequences of siRNAs used in this thesis.


2.2 Methods

2.2.1 Cell culture

Human osteosarcoma (U2OS) cells and cervical carcinoma (HeLa) cells

purchased from ATCC were maintained in Roswell Park Memorial Institute medium

(RPMI, Sigma-Aldrich) supplemented with 10% foetal bovine serum (Sigma-

Aldrich). Cells were grown in an environment containing 21% oxygen and 5% CO2

at 37°C. Cells were passaged by washing with Phosphate Buffered Saline (PBS),

incubating in trypsin-PBS solution for 5min and re-suspending in RPMI to neutralise

trypsin. Cells were then re-seeded at desired concentration.

2.2.2 Site directed mutagenesis

Mutations of SQ phosphorylation sites were performed using the Pfu-Ultra kit

(Stratagene) as directed by the manufacturer.

2.2.3 Transfection of plasmid

Mammalian expression vectors were transfected into cells using Lipofectamine 2000

(Life Technologies) or Fugene (Roche) as directed by the manufacturer. Briefly 5 μg,

10 μg and 15 μg of plasmid where used for T25, T75 and T175 flasks respectively. If

using Lipofectamine 2000, two eppendorf tubes were prepared with 250 μl of

Optimem (Life Technologies). In one tube the volume for the required concentration

of plasmid DNA (5 μg- 15 μg) was pipetted. In the second tube a volume of

Lipofectamine at a 3:1 ratio to plasmid amount was pipetted. The tubes were incubated

for 5 min, after which the two solutions were combined then allowed to incubate for

20 min before being transferred to the cells in flasks at 70-80% confluence.

Experiments were performed at 24-48 hr post transfection, as indicated. For Fugene


transfection, the Fugene reagent was incubated to room temperature before use. 100

μl of Optimem was pipetted into one eppendorf tube followed by the required volume

of DNA and lastly the Fugene reagent at a 1:3 ratio (1 μg of DNA and 3μl of Fugene).

After 20min incubation the solution was added to cell culture flask that contained cells

at 70-80% confluence.

2.2.4 siRNA mediated depletion of FKBP52

To study the function of FKBP52, esiRNA (Sigma-Aldrich) and silencer select (Life

Technologies) systems were used. esiRNA consists siRNA pools consisting of a

heterogeneous mixture of siRNAs that all target the same mRNA sequence. This

sequence is usually quite large and leads to highly specific and effective gene silencing

(Figure A1.4, in appendix A has FKBP52 sequence, and the esiRNA target in green).

Oligos used are listed in table 1). A non-specific sequence (GFP) for esiRNA or a

scramble sequence (silencer select) was used as control. Sufficient knock down

(>80%) was confirmed by western blot analysis. siRNAs were transfected using

RNAiMAX (Life Technologies) as per manufacture’s instructions.

2.2.5 Clonogenic survival assays

Clonogenic assays were performed as described previously (Paquet et al., 2015).

FKBP52 was depleted from cells by transfection of either esiRNA or silencer select

siRNA targeting the FKBP52 gene. 48 hr post transfection cells were either kept

untreated or treated with IR as indicated and subsequently seeded at a density of 400

cells per well into wells of a 6-well plate. Wells for three technical repeats were

prepared. Cells were grown for 10 days post treatment and then fixed and stained with

4% methylene blue (Sigma-Aldrich) dissolved in methanol. Colonies were counted


manually, and the surviving fraction at each dose was calculated (average number of

colonies counted/number of cells seeded) and expressed relative to untreated cells for

each data set. Each assay was performed at least 3 times.

2.2.6 Immunofluorescence microscopy

U2OS cells were seeded in a T25 flask the day before siRNA transfection. Following

siRNA, transfection cells were allowed to grow for 24 h before plating on to (IBIDI)

96 well Plate. Cells were allowed to grow for 24 hr before exposure to 6 Gy IR. Cells

were pre-permeabilized in 20 mM HEPES (pH 8), 20 mM NaCl, 5 mM MgCl2, 1 mM

ATP, 0.1 mM N2OV, 1 mM NaF and 0.5% NP40 for 5 min with the buffer kept at 4°C.

Following washes in PBS, cells were fixed in 4% paraformaldehyde for 20 min,

washed again with PBS before blocking with 5% BSA in PBS for 30 min. Cells were

then incubated with the appropriate antibody; Wells were washed with PBS and

incubated with secondary antibodies for 1 hr at ambient temperature before the nuclei

were counterstained with 4′, 6-diamidino-2-phenylindole hydrochloride (DAPI;

Sigma-Aldrich).

Images were collected on a Deltavision PDV microscope and collated for Figures with

Image J and Microsoft power point.

2.2.7 Immunoprecipitation

To detect an association between hSSB1 and FKBP52, immunoprecipitation from

U2OS cell lysates was performed using ice-cold 20 mM HEPES pH 8, 150 mM KCl,

10 mM MgCl2, 0.5 mM EDTA, 0.2% NP40, 0.5 mM DTT, 5% glycerol, 1 mM NaF,

1 mM NaVO4, protease inhibitor cocktail (Sigma-Aldrich).

Protein concentration was determined by bicinchoninic acid (BCA) assay (Sigma-

Aldrich). Equal amounts of lysate were incubated with the appropriate antibody or


isotype IgG overnight at 4°C with gentle agitation. Antibodies were captured using

magnetic protein A/G Dynabeads (Invitrogen), washed five times in NP40 buffer

containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors

(Cell Signalling Technology) prior to immunoblot analysis.

2.2.8 Flag precipitation

Anti-FLAG M2 magnetic beads were used to extract Flag constructs from protein

lysates. Lysates were prepared using IP buffer (see above). Beads were prepared by

washing them in IP buffer three times with gentle agitation at 4 °C. Once lysates were

prepared, 5 μl of beads were used per T175 flask of cell lysate. Lysates and beads were

incubated for 2 hr at 4 °C with gentle agitation. After incubation cells were washed

five times in 4°C IP buffer to remove any unbound proteins. Beads were eluted using

2X loading dye with 20% B-mercaptoethanol and incubated for 10 min at 80 °C.

Samples were then loaded on to a 4-12% SDS page gel as described, using a magnetic

rack to immobilise the magnetic beads.

2.2.9 Protein extraction

Protein was extracted from cells using Phospho extraction buffer or RIPA buffer. Cells

were harvested by trypsinisation or scraping, resuspended in PBS then spun down at

400 G for 10 min. PBS was removed, and lysis buffer with Protease inhibitors and

Phosphatase inhibitors was added. Cells were left on ice for 10 mins to allow lysis then

sonicated two times with 3 sec burst to degrade DNA. Once sonication was complete

cells were spun down at MAX speed at 4°C for 15 min, supernatant was removed and

ready for quantification or to be prepared to be run on an SDS page GEL.


2.2.10 Neutral Comet Assay

Cells were lifted by trypsinising cells for 5 mins at 37˚C immediately following mock

or ionising radiation treatment, and 103 cells were mixed with 0.6% low-melting point

agarose (Bio-Rad) (37°C in 1 X TBE). The cell suspension was spread onto a comet

slide (TREVIGEN) and immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10

mM Tris (pH10), 1% Triton X-100) for 30 min at 4 °C. Slides were immersed in TBE

for 15 min before electrophoresis at 70 volts (∼90 mA) for 30 min. Slides were then

washed in dH2O and immersed in 70% ethanol for 5 min and dried at 45°C for 15–30

min. The DNA was stained using SYBR® Green I (Sigma-Aldrich) (1:10 000) before

being dried completely and visualised using a Nikon Eclipse Ti microscope.

Quantitation of comet tail moments was performed on a minimum of 50 cells using

Image J plugin where the densitometry of the head and tail, as well as length, are

measured to calculate the comet tail moment.

2.2.11 Polymerase chain reaction (PCR) and agarose gel electrophoresis.

PCR reaction mixtures contained 10-100 ng of DNA template, 200 nM of

forward and reverse primer (see Table 1), 0.2 mM dNTP (Roche), 1x final

concentration Taq DNA polymerase buffer (supplied with polymerase), 1.25 units of

Taq DNA polymerase and nuclease-free H2O (total volume of 50 μl). Reactions were

performed using a Bio-Rad S1000 Thermal Cycler (Bio-Rad, Gladesville, Australia).

Cycling conditions were 95°C for 3 min, 20-35 cycles of 95°C for 30 sec, annealing

temperatures of 50-65°C for 30 sec (see Table 2.1 for specific primer temperatures),

68-70°C for 30 sec (Platinum Taq, 72°C; Pfu Ultra, 68°C; Expand High Fidelity DNA

polymerase, 72°C) and a final extension step of 68-72°C for 10 min. Reaction products

in 50 μl were mixed with agarose gel loading dye (see section 2.1.5) and


electrophoresed on 0.8-1% (w/v) agarose gels prepared with TAE (see section 2.1.5)

containing SYBR Safe (1:10,000 (v/v); Invitrogen). DNA molecular weight was

assessed using DNA Molecular Weight Marker X (0.07-12.2 kbp) (Roche).

2.2.12 Western blot analysis

Cells were lysed and sonicated. Lysates were cleared by centrifugation and protein

concentrations were estimated using the standard Bradford assay (Bradford reagent

supplied by Bio-Rad). Typically, 10 μg of protein lysate was separated on a 4–12%

sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel (Invitrogen). Gels

were run for 45min in MES buffer at 165V then transferred to a nitrocellulose blot via

40V for 1hr at 4°C in ATM transfer buffer and immunoblotted with the indicated

antibodies.

2.2.13 Homologous Recombination assay

A GFP reporter system was utilised to determine if FKBP52, like hSSB1, is involved

in the repair of double strand breaks by Homologous Recombination (HR). This can

be employed to look both at total homologous recombination. The MCF7-DRGFP

cells (HR reporter cells) contain DR-GFP, which is composed of two differentially

mutated green fluorescent protein (GFP) genes orientated as direct repeats (Litman et

al., 2005). The upstream repeat contains the recognition site for the rare-cutting I-Sce1

endonuclease, and the downstream repeat is a 5' and 3' truncated GFP fragment. These

cells were depleted of FKBP52 with siRNA, and these cells will be transfected with

an I-Sce1 expressing plasmid. This induces a break in the GFP reporter, which when

repaired by HR will result in a GFP signal that can be detected via fluorescence assisted

cell sorting (FACS) using the Gallios (Beckman Coulter)


2.2.14 DNA damage induction

Cells were treated by 6 Gy of gamma irradiation (IR) unless otherwise indicated, using

the Nordion Gammacell 40 irradiator which has a Caesium137 radiation source, or

using a Faxitron RX-650 cabinet X-ray irradiator (Faxitron Bioptics, Tucson, Arizona,

USA). Cells were harvested at the indicated time points post treatment.

2.2.15 Proliferation assay

Cells were transfected with either siRNA or plasmid or both as per previous

procedures. Wells were seeded at 5000 cells in a 96 well plate with 3 repeats per

sample. Cells were allowed to grow for 96 hr with images being taken via the Incell

2200 every 24 hr to measure the cell density. Image files were analysed via Cell

Profiler software to determine cell area every 24 hr.

2.2.16 Annexin PI staining

Annexin PI staining was performed as per kit instructions (ENZO) in summary,

Binding Buffer (4X) was diluted 1:4 in distilled water (50ml binding buffer and 150ml

distilled water). Cells were washed in PBS by gentle shaking or pipetting up and down.

Cells were Resuspended in Binding Buffer (1X). Cell density was adjusted to 2-

5x105/ml, and 5 μl Annexin V-FITC was added to 195 μl cell suspension. Cells were

mixed and incubate for 10 min at room temperature. Cells were then washed and

resuspended in 190 μl Binding buffer (1x) and10 μl Propidium Iodide (20 μg/ml) was

added. Cells were then analysed via flow cytometry.

2.2.17 Protein purification

Proteins were expressed in E. coli Bl21 shuffle strain, and cells were grown at 30 oC

to an Optical Density of 0.6 and protein expression was induced using 0.4mM IPTG


for 12 hours at 16 oC. Frozen pellets were sonicated in cell lysis buffer (50 mM Tris–

HCl (pH 7.5), 10% sucrose, 10 mM EDTA, 600 mM KCl, 0.01% Igepal CA-630

(Sigma-Aldrich), St. Louis, MO), 1 mM dithiothreitol (DTT) in the presence of protein

inhibitors (chymostatin, leupeptin, aprotinin, and pepstatin, at 2 mg/ml each). The cell

lysate was ultracentrifuged at 45 k rpm for 1 hr.

In the case of the maltose-binding protein tagged hSSB1 (MBP-hSSB1), the

clarified supernatant was resolved on a 40 mL SP sepharose fast flow (GE Healthcare)

column using an AKTA FPLC (GE Healthcare) with a 5 column volume gradient of

100 to 1000 mM KCl in buffer K (20 mM KH2PO4, pH 7.4, 0.5 mM EDTA, 10%

glycerol, 0.01% Igepal CA-630, 1 mM DTT). Fractions containing hSSB1 were pooled

and incubated with Amylose resin (New England Biolab) for 2 h at 4°C. Following

extensive washes, amylose-bound protein was eluted with buffer K containing 10 mM

Maltose and 300 mM KCl. Fractions containing hSSB1 were pooled, concentrated

using a 30 kDa cut off Amicon ultra centrifugal device (Millipore) to a volume of 250

µL and loaded on a Superdex200 10/300 GL size exclusion chromatography column

(GE Healthcare) run with K buffer containing 300 mM KCl.

His-FKBP52 was purified following the same protocol, with fractions from the

SP column that contained FKBP52, incubated with 10 mM imidazole and Ni-NTA

agarose (Qiagen) for 2 h at 4°C. Following extensive washes, Nickel-bound protein

was eluted with buffer K containing 200 mM imidazole and 300 mM KCl. Pooled and

concentrated protein was then subjected to size exclusion chromatography

Protein concentrations were estimated by running a dilution series on SDS-

PAGE gel.


2.2.18 Size exclusion- Multi-angle light scattering (MALS)

Size exclusion chromatography coupled to multi-angle laser light scattering

was carried out by injecting 250 µL (corresponding to 250 µg) of purified FKBP52 in

MALS buffer (20 mM Tris pH 7, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) onto a

Superdex 200 10/300 GL size exclusion column mounted to an AKTA

chromatography system in tandem with Viscotek SEC-MALS 20 (Malvern/ATA

scientific). Data was collected and analysed with the OmniSEC software.

2.2.19 Direct protein interaction assay

Pull down assays were conducted by incubating 2 μg of his-FKBP52 with or

without 2 μg of MBP-hSSB1 for 30 min on ice. After addition of 10 μl of amylose

resin, the mixture was further incubated at 4°C for 2 hr, under light agitation. After

centrifugation at 7000rpm for 1 min, the supernatant (S) was collected, and beads were

washed 3 times with K buffer complemented with 100mM KCl. After the final wash,

the beads were resuspended in 20 μl of SDS-Page loading buffer. Supernatant (S),

wash 3 (W) and Elution (E) were resolved on SDS-Page gel and visualised by

coomassie blue staining.

2.2.20 qRT-PCR

Quantitative real time PCR (qRT-PCR) reactions were performed in 384 well

plates (Axygen) and contained l µl diluted cDNA reverse transcribed from total RNA

(equivalent to 0.5 g) in nuclease-free H2O (1:5), 50 nM forward and reverse primer,

1x final concentration of SYBR green PCR master mix (Applied Biosystems) and

nuclease-free H2O (total volume of 10 µl). Reactions were performed using a ViiA7

real-time PCR system (Life Technologies). Cycling conditions were 95 ◦C for 10 min,

40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min followed by a primer-template


dissociation step. Gene expression was normalised to 7SL mRNA levels using the

comparative CT (CT) method (Adams, Pagel, Mackie, & Hooper, 2012; Paquet et al.,

2015). The primer sequences for the human genes are in Table 1.

2.2.21 Cell cycle synchronisation double Thymidine block

Cells were plated at 30% confluency in T25 tissue culture flasks for each time

point of the experiment and allowed to adhere to the flask. This allowed me to track

the progression of the cell cycle over a 24 hr period. Thymidine was added to the

flasks at a final concentration of 2 mM and allowed to incubate in 37 °C cell culture

incubator for 16-18 hrs. After incubation Thymidine media was removed and cells

were washed with PBS then allowed to grow for 6 hrs with fresh media. After 6 hrs

thymidine was added to flask for a final concentration of 2 mM and cells were left to

incubate in tissue culture incubator at 37 °C for 16-18 hrs. After incubation cells

were released via a 1X PBS wash and fresh RPMI media was added to cells then

cells were placed in the incubator. Cells were harvested at 0 hr, 4 hr, 8 hr, 12 hr, 16

hr, 20 hr and 24 hr post release via cell scrapping and stored at -80 °C or were stored

in 70% ethanol 30% PBS at -20 °C for Flow cytometry analysis. ethanol was added

during agitation via a vortex and ethanol was at 4 °C.

2.2.22 Gene editing utilising CRISPR

To fully deplete FKBP52 from U2OS and Hela cells CRISPR was used as

described by F Ann Ran et al., (2013). Briefly, CRISPR technology allows for the

targeted mutation of genes in the genome and can be used to change the protein

sequence. Identification of CRISPR sites was performed using CRISPR sgRNA (sub

genomic RNA) design tool (crispr.mit.edu). Identified sites were targeted with primers


designed and integrated into pSpCas9n(BB)-2A-Puro (Addgene plasmid ID: 48141)

(Ran et al., 2013) using the BbsI restriction enzyme, with CACC at 5’ of the top oligo

and AAAC at the 5’ of the bottom oligo for overhangs. A 3’ C nucleotide was added

for effective insertion onto the bottom oligo. As per the protocol published by (Ran

et.al 2016), positive colonies were selected via puromycin selection and sequenced to

confirm sequence integration. Plasmids with the integrated FKBP52 targeting plasmid

were transfected into cells. Cells were seeded to form single colonies and allowed to

proliferate until 50% confluent to allow FKBP52 knockout screening using western

blotting. FKBP52 depleted cells were selected and expanded to assess the effect of

FKBP52 depletion.

2.2.23 MG132 Protease inhibition

Cells were plated at 30% confluency and allowed to adhere to cell culture flask.

MG132 was added to flasks at a final concentration of 5 µM and cells were placed in

incubator for 8 hrs. Cells were harvested via trypsinising and processed for western

blotting.

2.2.24 Sodium butyrate HDAC inhibitor

Cells were plated at 30% and allowed to adhere to cell culture flask. Sodium

Butyrate was added to flasks at a final concentration of 10 mM and cells were placed

in incubator for 8 hrs. Cells were harvested via trypsinising and processed for western

blotting.


2.3 Statistics

Results are mean ± SEM of at least 3 independent experiments unless otherwise

stated. Statistical significance was assessed by Student’s t test. P < 0.05 was

considered significant.

Chapter 3: Investigating the interaction between FKBP52 and hSSB1 45

Chapter 3: Investigating the interaction between FKBP52 and

hSSB1

3.1 Introduction

Human single stranded DNA binding protein 1 (hSSB1) plays a crucial role in the

maintenance of the genome and responding to DNA damage (Richard et al., 2008; Y.

Wu et al., 2015). Following DNA damage hSSB1 relocates to DNA damage sites

where it is phosphorylated by ATM. hSSB1 can also change the kinase activity of

ATM and the ATM-dependant cell cycle check point activation (Richard et al., 2008).

It has also been shown that, hSSB1 is required for efficient recruitment of the MRN

complex to DNA damage sites and binds directly to NBS1 from this complex (Richard,

Cubeddu, et al., 2011a; Richard, Savage, et al., 2011b). Furthermore, Y. Wu et al.,

(2015) have presented data showing that hSSB1 plays a crucial role in the cell cycle

and response to DNA damage by modulating p53 and p21. However, little is known

about the regulation of hSSB1. In this chapter, I explore a potential relationship

between FKBP52 and hSSB1. FKBP52 has been shown to be involved in the nuclear

transport and stability of proteins involved in the maintenance of the genome such as

NF-κB, hTERT and p53. With the role of FKBP52 in mind and the data from a

connectivity screen, I explore the possibility that FKBP52 and hSSB1 may share a

similar relationship.

3.2 Validation of antibodies and siRNA

In order to study FKBP52, antibodies and siRNA were first validated. Two

commercially available antibodies were tested to detect FKBP52 by western blotting:

rabbit polyclonal anti-FKBP52 (Sigma-Aldrich Prestige), and mouse monoclonal anti-

FKBP52 Hi52C (Abcam). The specificity of the two antibodies were tested via lysates


depleted of FKBP52 using siRNA. As shown in Figure 3.1a and b both antibodies

specifically detect the FKBP52 protein at the predicted molecular weight of 52 kDa.

However, the mouse anti-FKBP52 Hi52C had a stronger affinity for a non-specific

band than for FKBP52 and required a 1/500 dilution, while the rabbit anti-FKBP52

(Sigma-Aldrich Prestige) predominantly detected FKBP52 and required a 1/5000

dilution to detect FKBP52 in protein lysates. It was thus decided to use the anti-

FKBP52 Sigma Prestige antibody for western blotting.

Also of interest was the effect of the siRNA on the proteins in this blot. Silencer select

was utilised for the antibody optimisation experiment, and it can be seen that the larger

band detected has not been affected by the siRNA, indicating it is a nonspecific

product. However, the lower band detected by the Sigma prestige antibody appears to

be depleted by the siRNA indicating it could be another form of FKBP52, such as a

cleaved form.

Next, the two antibodies against FKBP52 were tested for use in

immunofluorescence (IF) microscopy. U2OS cells transfected with FKBP52 or control

siRNA were fixed and probed with the Sigma-Aldrich FKBP52 antibody (Figure 3.1

c and d). It was observed that FKBP52 localised in the cytoplasm and the nucleus of

non-extracted cells (Figure 3.1 d). This was consistent with previous results that

showed FKBP52 to occur throughout the cell (Sanchez, 1990).


Figure 3.1 Optimisation of reagents. Comparison of FKBP52 antibodies. Cells were treated with siRNA targeting FKBP52 or a scramble control (siCtrl), protein was extracted and ran on a 12-4% SDS page gel and transferred to a nitrocellulose membrane. The first blot a) is the Sigma antibody only in grey scale. The second blot b) is the Abcam antibody only in grey scale. Immunofluorescence staining of FKBP52 c) Immunofluorescence of U2OS cells that have been treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were fixed using 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus. d) Immunofluorescence of U2OS cells that have been treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were pre-extracted to remove -soluble cellular proteins prior to fixation in 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus. e) And f) Depletion of FKBP52 using esiRNA e) and silencer select siRNA f). Western blots of U2OS cells transfected with esiRNA targeting FKBP52 or an esiRNA targeting the green fluorescence protein GFP as a control. Cells were harvested at 48,72 and 96 hr post transfection


To investigate whether FKBP52 localised to the chromatin, cells were treated

with an extraction buffer to remove soluble proteins as used previously Richard et al.,

2008. In pre-extracted cells the levels of FKBP52 were reduced in both the cytoplasm

and nucleus (Figure 3.1 d), suggesting that a proportion of FKBP52 exists in a soluble

form in both the nucleus and the cytoplasm of the cell, and a proportion of FKBP52 is

bound to the cytoskeleton and chromatin within the cell. The use of siRNA validated

the specificity of the FKBP52 antibody in immunofluorescence as shown by greatly

reduced staining levels in both the pre-extracted and non-extracted FKBP52 depleted

cells. The Hi52C monoclonal antibody was also tested and was found to produce too

much background signal to be useful for IF procedures.

Throughout the project, two types of siRNA against FKBP52 were used: esiRNA

(Sigma-Aldrich) and silencer select (Thermo Fisher). The esiRNA is a heterogeneous

mixture of 21 bp oligos covering a large section of the target gene sequence. This

method was the initial method chosen as it had been previously shown in the lab to

produce effective and persistent depletion of FKBP52. However, as esiRNA contain a

pool of different sequences they cannot be used in rescue experiments, which require

expressed constructs to be resistant to depletion with a single sequence of siRNA. For

this reason, a sequence-defined siRNA sequence was also chosen. The defined

sequence FKBP52-targeted siRNA was in the form of “silencer select” from Thermo

Fisher, which is a locked nucleic acid chemistry that is thermo-stable and has a greater

affinity to the target site than standard siRNA technologies (Elm n, 2005). Silencer

select was also used due to its successful use to target other proteins of interest within

the CARP laboratory at QUT. Both types of siRNA types were shown to be efficient

at decreasing the FKBP52 protein levels up to 96 hr post transfection (Figure 3.1 e and

f). However, at the maximum recommended concentration of 10 nM, silencer select


appears to knock down FKBP52 protein faster and more efficiently than esiRNA

(Figure 3.1 f). Both siRNA methods were used throughout the project as indicated. A

more detailed optimisation of siRNA and antibodies is provided in Appendix A.

3.3 hSSB1 and FKBP52 are part of the same complex in cell lysates

Upon identification of FKBP52 as a gene co-regulated with hSSB1, I next sought

to determine if an interaction between the two proteins in a DNA damage context could

be identified.

Co-immunoprecipitation (co-IP), a commonly used method to study protein-

protein interactions in cell lysates was performed. To avoid the IgG interference with

FKBP52 at 55 kDa, the hSSB1 antibody was cross-linked to the Protein G Dynabeads.

The beads were incubated with the lysate and the immune complexes were then

separated on SDS-PAGE gels and western blotted for hSSB1 and FKBP52. As shown

in Figure 3.2 a, FKBP52 co-immunoprecipitated with hSSB1 at 1 and 2 hrs after

induction of double strand DNA breaks by IR. This suggests that FKBP52 and hSSB1

may form a complex in cells, in response to DSB induction. The reverse co-IP was

performed using anti-FKBP52 linked to protein A beads. However, hSSB1 was not

detected in this reaction (Figure 3.2 b). Interestingly HSP90, a known interactor of

FKBP52 was shown to interact with FKBP52 in an IR responsive manner. The HSP90

and FKBP52 proteins have been shown to interact in the co-chaperone mechanism in

the maturation and transport of hormone receptors, but to date, no evidence exists that

this interaction increases in a DNA damage context. The lack of FKBP52:hSSB1

interaction could be explained by masking of the interaction site by the antibody

epitope or that the level of interaction was below the detection level of this assay.


Figure 3.2 Co-Immunoprecipitation of FKBP52 and hSSB1. U2Os cells were exposed to 6 Gy of IR and harvested 1,2,4 and 6 hours post IR and an Untreated control was included. a) Co-IP of hSSB1. Anti-hSSB1 goat was cross linked to dyna beads then incubated with cell lysates to remove hSSB1 from solution and any proteins bound to hSSB1. These beads were removed from the solution washed, and boiled to remove hSSB1 from the beads. Samples run on SDS-Page gel and probed with anti-FKBP52 rabbit (Sigma) and hSSB1 goat. b) Anti-FKBP52 rabbit (sigma) was incubated with dyna beads and cell lysates to remove FKBP52 from solution and any proteins bound to FKBP52. These beads were removed from the solution washed, and boiled to remove hSSB1 from the beads. Samples were run on SDS-Page gels and probed with anti-FKBP52 rabbit (sigma) and hSSB1 goat and anti HSP90 goat (abcam). Time points in hours are indicated above lanes as numerical values.


3.4 FKBP52 and hSSB1 do not interact directly in vitro

To explore further the FKBP52 and hSSB1 interaction, FKBP52 and hSSB1 were

purified in collaboration with Dr Nicolas Paquet from our laboratory. FKBP52 was

expressed in E. coli BL21 shuffle strain from the pet28a plasmid. hSSB1 was purified

by ion exchange chromatography followed by affinity purification using a Ni-NTA

column and then finally using a size exclusion column as illustrated in Figure 3.3 a.

This demonstrated that FKBP52 was present both as a dimer and as a monomer in

solution (Figure 3.3 c). To explore a direct interaction, hSSB1 was attached to

cyanogen bromide activated agarose and passed over the purified recombinant

FKBP52. As can be seen in Figure 3.3d FKBP52 did not appear to have a stable

interaction with hSSB1. This may indicate that hSSB1 itself is not a target of FKBP52

or that the interaction requires hSSB1 to be in an unfolded state and/or either FKBP52

or hSSB1 require post-translational modifications for their interaction. One other

possibility is that this data could indicate that the interaction is not a direct interaction

and it is mediated by another protein or complex.


Figure 3.3 FKBP52 and hSSB1 do not interact directly a) Schematic of FKBP52 purification strategy. Cell lysate was centrifuged at 100,000 rpm, and loaded on a SP column. Fraction containing FKBP52 were incubated with Ni-NTA resin and following elution the protein was subjected to a size exclusion chromatography b) Recombinant his-FKBP52 1. 2 μg of recombinant FKBP52 were resolved on non reducing SDS-Page gels, and stained using coomassie blue c) FKBP52 exist in dimers in solution. FKBP52 proteins (~200 µg) were applied to a Superdex 200 column with an in line MALLS detector to determine weight-averaged molecular weight in solution. The elution (continuous line) and light-scattering (▪) are shown. d) 2 μg of MBP-hSSB1 and 2μg of his-FKBP52 were incubated overnight at 4˚C in K buffer containing 75 mM KCL, and trapped using amylose resin The beads were washed and treated with SDS to elute the bound proteins. The supernatant (S), wash (W), and SDS elute (E) were analysed by SDS-PAGE and stained by Coomassie blue G250.

a

c

b

d


3.5 FKBP52 depletion abrogates hSSB1 nuclear localisation

FKBP52 has been shown to be required for the modification and shuttling of

proteins from the cytoplasm to the nucleus. This role has been documented to occur in

a co-chaperone mechanism with steroid receptors and independently with the protein

NF-κB p65 (Erlejman et al., 2014; Lagadari et al., 2015; Storer et al., 2011). With this

being the canonical role of FKBP52 and data showing a potential interaction of

FKBP52 and co-regulation with hSSB1, the potential that FKBP52 could alter the sub

cellular localisation of hSSB1 was investigated. U2OS cells were depleted of FKBP52

using siRNA then seeded into 96 well plates and allowed 24 hrs to attach. Cells were

then treated with 6 Gy IR to induce a DNA damage response. Before fixation, cells

were pre-extracted to wash away soluble proteins allowing easier visualisation of

proteins bound to the chromatin. These cells were incubated with FKBP52 and hSSB1

antibodies to determine if colocalisation occurred and if hSSB1 levels and localisation

in the cell had been affected by FKBP52 depletion.

As previously shown by the Richard Lab, in response to IR, like other DNA

repair proteins hSSB1 binds to the chromatin rapidly forming foci at sites of DNA

damage (Richard, Savage, et al., 2011b). When cells have been depleted of FKBP52,

less hSSB1 foci appear to form in the nucleus in response to IR and there is a higher

level of non-nuclear hSSB1 (Figure 3.4 a). This reduction in hSSB1 nuclear levels has

been quantified using the Incell 2200 system (Figure 3.4 b) and shows a 0.5-1.5 fold

decrease in nuclear hSSB1 levels in FKBP52 depleted cells. This reduction in nuclear

foci could indicate that FKBP52 is required for the formation of hSSB1 foci,

potentially via transporting hSSB1 to the nucleus or modifying hSSB1 to activate the

protein.


Figure 3.4 FKBP52 and hSSB1 immunofluorescence and cellular localisation after IR a) Immunofluorescence of cells with endogenous (siCtrl) or depleted FKBP52 (siFKBP52) cells were exposed to 6 Gy IR then pre-extracted and fixed at indicated times. Cells probed with DAPI (blue), anti-FKBP52 (red) and anti-hSSB1 (green) antibodies. b) InCell2200 analysis of cell nucleus immunofluorescence intensity comparing siCtrl and siFKBP52 population. Data is graphed as mean +- standard error of the mean from 2 independent experiments. T tests were used to compare siCtrl and siFKBP52. ns = > 0.05,* = P < 0.05, ** = P < 0.01, *** = P < 0.001. P values relate to the nuclear intensity of hSSB1 in control and FKBP52

Chapter 4: FKBP52 and its role in DNA damage response 55

Chapter 4: FKBP52 and its role in DNA damage response

4.1 Introduction


with tens of thousands of DNA lesions being produced per day per cell. If DNA

damage is not repaired correctly, it can be detrimental to the cell, leading to alterations

in the genetic material and disease states such as cancer and neurodegenerative

disorders. To protect the integrity of the DNA, cells have evolved a global signalling

network known as the DNA damage response. This network detects different types of

genotoxic stress to raise a versatile and coordinated response, which includes control

of the cell cycle transitions, transcriptional processes and stimulation of DNA repair

and apoptosis. The aim of this Chapter is to establish if FKBP52 has a role in this

process. With previous data indicating that FBKP52 depletion prevents hSSB1

accumulation to the nucleus, I wanted to establish if FKBP52 responds to DNA

damage events and if depletion or over expression of FKBP52 has an effect on known

DNA damage pathways.

4.2 The FKBP52 is destabilised in response to IR and localises to the nucleus

hSSB1 functions in a number of DNA repair pathways. However, the best

characterised to date is its role in the repair of double strand DNA breaks by

homologous recombination (Y. Li et al., 2009; Richard et al., 2008). To determine if

FKBP52 responds to DNA double strand breaks U2OS cells were exposed to 6 Gy IR

and harvested at 1 hr, 2 hr, 4 hr and 6 hr post treatment and lysates subjected to western

blot analysis. A band at 52 kDa, the predicted FKBP52 size, was observed (Figure


4.1). This band initially decreases in intensity after IR treatment before retuning to

basal levels by 6 hr. The lower molecular weight FKBP52 band also fluctuates in

intensity over this 6 hr period and appears to increase in intensity as the 52 kDa band

decreases in intensity. This could potentiallys indicate cleavage of the FKBP52 protein

in response to DNA damage. To exclude the possibility that the lower band could be

the homologous FKBP51, a western blot was performed with anti FKBP51 antibodies.

However, no reactivity to the FKBP51 antibody was detected with this band, indicating

as before that this was likely FKBP52. However, when investigating the epitope of

both antibodies, they appear to target a similar highly conserved sequence of the

FK506 immunophilin family. These antibodies share 46% epitope sequence similarity,

indicating there could be difficulties in differentiating these proteins with the chosen

antibodies. Additionally, there appears to be a weak signal at the approximate size of

the 44 kDa band. This could be due to the similarity of the antibody sequence or

potentially FKBP51 could also be producing a smaller cleavage product.


Figure 4.1 FKBP52 response to IR. a) Cells were exposed to 6 Gy IR then protein lysates harvested at 1, 2, 4 and 6 hours post IR and an untreated (Unt) control, and run on SDS page gel and western blotted. Blots were probed with anti-FKBP52), anti-FKBP51 and anti-β-actin antibodies. b) Densitometry of FKBP52 full length and 44 kDa bands after exposure to 6 Gy IR.


FKBP52 shows a response to induction of double strand DNA breaks by IR

when analysed by western blot. After IR there is the appearance of a lower migrating

(44 kDa) form of FKBP52 as well as the initial reduction in the full length FKBP52. I

also observed that FKBP52 formed foci with similar kinetics to hSSB1 in Figure 4.2.

To further investigate the FKBP52 response following induction of DNA breaks by

IR, the localisation of FKBP52 within the cell was investigated by

immunofluorescence microscopy. It was of interest to examine whether, like hSSB1,

FKBP52 also localises to sites of DSB on the chromatin following pre-extraction prior

to fixation. As mentioned previously, this method removes all soluble proteins from

the cell, leaving only chromatin and cytoskeletal bound proteins. Many DNA repair

proteins have altered cellular localisation following induction of DNA damage.

Indeed, hSSB1 becomes chromatin bound after IR treatment. It was found that

FKBP52 levels increase both in the cytoplasm and the nucleus at 2 hr post IR treatment

(6 Gy) (Figure 4.2). FKBP52 chromatin bound levels then decrease at 4 hr and increase

again at 6 hr. The cytoplasmic localisation of FKBP52 likely represents cytoskeletal-

bound FKBP52, in agreement with published data showing FKBP52 interacts with

cytoskeletal bound dynein motor proteins (Galigniana, Echeverría, Erlejman, &

Piwien-Pilipuk, 2014; Harrell et al., 2004; Storer et al., 2011). FKBP52 also appears

to increase in level in the nucleus but not the nucleoli suggesting that it is bound to

chromatin.

Following induction of double strand DNA breaks a histone marker is seen to

rapidly accumulate at the break site. This marker referred to as γH2AX, is often used

as an indirect marker of the site of the double strand DNA break. Using

immunofluorescence of U2OS cells we observed distinct foci of FKBP52 suggesting


Figure 4.2 Subcellular localisation of FKBP52 in response to IR. Immunofluorescence microscopy of U2OS cells that have been irradiated with 6 Gy IR. Cells were pre-extracted to remove soluble cellular proteins then fixed using 4% PFA. Cells were probed with antiFKBP52 and γH2AX antibodies and DAPI was used to stain the nucleus (blue).


that FKBP52 may be at the sites of double strand DNA breaks. However, these sites

did not co-localise with γH2AX (an epigenetic marker of break sites). This either

suggests that FKBP52 does not sit at sites of double strand DNA breaks and/or is

recruited to sites of these breaks with different kinetics to γH2AX. Another possibility

is that FKBP52 sits proximal to the break site. Indeed, Figure 4.2 demonstrates

different recruitment kinetics of γH2AX and FKBP52, with γH2AX being recruited

early and resolved by 4 hr while maximum recruitment of FKBP52 does not occur

until 6 hr post IR treatment suggesting a temporal difference between FKBP52 and

γH2AX.

To further confirm the levels of FKBP52 in different compartments of the cells

in response to IR, sub cellular fractionation combined with western blotting was

performed. This involves using different buffers and centrifugation steps to separate

the cell pellet into a cytoplasmic soluble fraction, membrane fraction, soluble nuclear

fraction, chromatin bound fraction and the micrococcal nuclease resistant chromatin

fraction. The three fractions of particular interest were cytoplasmic, soluble nuclear

and chromatin, this was due to previous literature showing FKBP52 as a nuclear

transport protein as well as proteins involved in DNA damage repair tend to bind to

the chromatin. The results indicate that cytoplasmic FKBP52 levels appear to remain

the same until 4 hrs (Figure 4.3). In the soluble nuclear fraction, FKBP52 appears to

increase in levels at 1 hr after IR to then go back to basal levels by 2 hr this appears to

coincide with the cytoplasmic levels, which follows the described model of FKBP52

using the cytoskeleton to shuttle from the cytoplasm to the nucleus. FKBP52 was not

found in the chromatin bound fraction but it was found in the micrococal nuclease

resistant fraction which could indicate FKBP52 bound to the tightly bound chromatin

after IR which hints towards FKBP52 being involved in the DNA damage repair


pathway. Though there is a difference between the results shown by the IF and the sub

cell fractionation. In the sub cell fractionation FKBP52 levels at the 4 hr time point

continue to be present at a constant level. However, when we look at the same time

point using IF, FKBP52 levels are seen to decrease at the 4hr time point and then return

to basal levels at 6 hrs.


Figure 4.3 Sub-cellular localisation of FKB

P52 in response to DN

A dam

age Sub cellular fractionation of U2O

S cells follow

ing exposure to 6 Gy of IR

then harvested at the indicated time points using a sub cellular fractionation kit. The

fractions shown are: C

ytoplasmic, M

embrane bound Soluble nuclear and C

hromatin and M

icrococcal nuclease resistant chrom

atin fraction. Fractions were probed w

ith anti-Nucleoloin, anti-FK

BP52, anti-actin and anti-H

3 antibodies.


4.3 FKBP52 levels are highest during the G1 phase of the cell cycle.

The data previously (section 4.2) generated have determined that FKBP52

responds to DNA damage caused by IR, indicated by changes in FKBP52 levels and

localisation. DNA repair proteins can be regulated during the cell cycle depending on

what process they are involved in. For instance, while NHEJ is performed throughout

all phases of the cell cycle, HR is only performed when a sister chromatid is present

and thus only occurs primarily in late S phase and G2. Cell cycle synchronisation

experiments were conducted to determine if FKBP52 levels fluctuate during the

different stages of the cell cycle. Cells were synchronised using the double thymidine

block (as described in the Method section), which arrests cells in the G1/S phase. Cells

were then released and harvested every 4 hr post the second thymidine addition.

Samples were harvested for both western blots and flow cytometry to determine the

phase of the cell cycle (Figure 4.4 c) at the time of harvest. As shown in Figure 4.4 an

analysis of the western blot demonstrates that FKBP52 levels fluctuate throughout the

cell cycle and are low during S and G2 phases of the cell cycle and highest in G1 (to

visualise the 44 kDa band the western blot had to be over exposed).


Figure 4.4 FKBP52 levels during the cell cycle a) western blot from cells treated with thymidine to synchronise cells in the G1/S phase. Cells were released and harvested every 4 hr for 24 hr. b) densitometry of western blot FKBP52 upper band normalised to action levels. c) Flow analysis of cells and percentage of cells in G1, S or G2 phase.


4.4 FKBP52 depletion leads to up regulation of certain DDR proteins

FKBP52 is a Proline Isomerase that acts in concert with Hsp90 in the correct

folding of target proteins. It has been shown that inhibition of Hsp90 function leads

to a reduction in protein levels of several DNA repair proteins. Therefore, the effect of

FKBP52 on DNA damage signalling/repair pathways was investigated.

U2OS cells were depleted of FKBP52 using siRNA and protein levels and

phosphorylation status of known DNA damage response proteins including BRCA1,

ATR, ATM, p53, CHK2 were investigated by western blotting (figure 4.5).

Interestingly, phosphorylation of BRCA1 at Serine1524, p53 at Serine-15 and P-ATM

at Serine 1981 and H2AX (γH2AX) appear to increase or are prolonged over a 6 hr

period when FKBP52 is depleted. This prolonged phosphorylation potentially

indicates that the DNA damage repair is incomplete, as γH2AX is a histone marker of

DNA breaks and ATM is phosphorylated when being recruited to the break site. p53

phosphorylation in response to DNA damage could indicate p53 is activating its

activities such as apoptosis, cell cycle arrest and cellular senescence, all important in

DNA repair. These results suggest that the cells are not undergoing proper repair when

FKBP52 is depleted. hSSB1 levels appear to increase in FKBP52 knockdown cells

indicating a potential role of FKBP52 in the regulation of the homologous

recombination pathway. It is possible that more DNA damage has occurred or

potentially that less repair is occurring when FKBP52 is depleted from cells. Total

protein levels of ATM likewise appear to be affected with an apparent decrease when

FKBP52 is depleted, as do Rad51 protein levels. As mentioned in Chapter 1 ATM is


Figure 4.5 Effect of FKBP52 depletion on DNA damage proteins a) western blot of U2Os cells that have been treated with siControl or siFKBP52 for 72 hr, exposed to 6 Gy IR and harvested at 1, 2, 4 and 6 hours post IR Cells were lysed and proteins harvested and ran on an SDS page gel then transferred to a western blot. westerns were probed with the above antibodies to determine protein levels.


important for the phosphorylation of yH2AX which in turn initiates a signalling

cascade that results in the recruitment of proteins involved in repair and the triggering

of cell cycle arrest. RAD51 functions to promote strand invasion of a sister chromatid,

(see pp4-7 for more detail.) This is reminiscent of a Hsp90 knockdown where Rad51

levels are depleted. Hsp90 levels appear unaffected which was expected as FKBP52 is

required for the co-chaperone complex and modification of proteins with HSP90, but

FKBP52 loss itself does not affect Hsp90 expression or stability. What is of interest is

that NF-κB p65 levels appear unaffected, in contrast, it has been shown previously that

FKBP52 affects the transport of NF-κB to the nucleus and the transcription in an

Hsp90 independent manner. NF-κB describes a group of transcription factors that

activate when certain stress occurs such as IR UV light and Reactive oxygen species.

The data presented here suggests that FKBP52 has a role in the DNA damage

response with depletion of FKBP52 prolonging the activation of DNA damage

markers (γH2AX) and proteins involved in DNA damage repair. The exact role

FKBP52 in response to DNA damage needs to be investigated further.

4.5 FKBP52 depletion using siRNA leads to radiosensitivity

In order to investigate the long-term effects of FKBP52 in irradiation-induced cell

death, clonogenic survival assays were performed. This assay is one of the gold

standard assays to predict potential radio-sensitising effects in vivo. U2OS cells were

either transfected with FKBP52 or control esiRNA. At 48 hrs post transfection, cells

were seeded at 400 cells/well (described in materials and methods), irradiated at 0, 2,

4, and 6 Gy and cultivated for 8 days. The number of colonies was then counted and


Figure 4.6 The effect of FKBP52 levels on cell survival after DNA damage a) Clonogenic assay of esiRNA cells treated with IR b) lysates of cells treated with esiRNA targeting FKBP52 densimotry value is written above lane to demonstrate knockdown. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siCtrl and siFKBP52. ns = > 0.05. Densitometry is indicated above each lane.


the surviving fraction was determined by normalising to the number of colonies in the

untreated samples. As shown in Figure 4.6 a, irradiation reduced the clonogenicity of

FKBP52 depleted U2OS cells in a dose dependent manner. The data is the average of

three independent experiments. This indicates that FKBP52 may have a radio-

sensitising effect on cells, in particular at doses above 4 Gy. Western blots were

performed to determine the level of knock down. As shown in Figure 4.6 b FKBBP52

was depleted to 20% of control cells (esiRNA).

Next, a potential effect of FKBP52 overexpression on long term radiation sensitivity

was investigated using the clonogenic survival assay. At 24 hrs post transfection of

either the pCMV6-FLAG or pCMV6FLAG-FKBP52 plasmids, U2OS cells were

seeded at 400 cells/well into 6 well plates. 24 hrs later, cells were irradiated at 0, 1, 2,

4 and 6 Gy and cultivated for 8 days. Colonies were counted and

surviving fractions were calculated by normalising to the untreated wells. As shown

in Figure 4.7a there was no difference in radiosensitivity between cells with

overexpression of FKBP52 compared to cells with endogenous levels of FKBP52.

Overexpression of FKBP52 was verified by western blotting (Figure 4.7b). However,

it should be noted that over-expression of FKBP52 itself caused a non-significant

reduction in the number of colony forming units suggesting either over-expression was

causing cell death or impairment in growth or replication (Figure 4.7c).


Figure 4.7 Over expression of FKBP52 and radiosensitivity a) Clonogenic survival assay of cells transfected with PCMV6-FLAG vector or PCMV6-FLAG FKBP52 vector. b) western blot presenting cells transfected with PCMV6-FLAG vector or PCMV6-FLAG FKBP52 vector, blot was probed for actin and FKBP52 c) Untreated well of Clonogenic assay was counted and normalised presented as percentage of surviving colonies. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns.


4.6 FKBP52 overexpression has no effect on apoptosis

It was observed in the clonogenic assays that cells overexpressing FKBP52 had

fewer cell colonies at 8 days post plating (Figure 4.7 c), indicating that overexpression

of FKBP52 might negatively affect cell viability. To determine if cell death was

occurring in FKBP52-overexpressing cells, annexin V labelling, a widely used assay

for quantification of apoptosis and necrosis was used. Following annexin V staining,

cells were analysed by FACS (Figure 4.8 and 4.9). In both Hela and U2OS cells, over

expression of FKBP52 appeared to have a modest affect, with an increase in live cells

when FKBP52 is over expressed and a decrease in apoptosis and necrotic cells. Figure

4.8 b presents the “dead” population and shows a decrease in apoptotic, preapoptotic

and necrotic Hela cells. FKBP52 overexpression had a much less pronounced effect in

U2OS cells (Figure 4.9 b) indicating a potential difference in the two cell types. This

may suggest that FKBP52 may protect cells from apoptosis and given the upregulation

of FKBP52 in cancer this may have implications for cancer treatment.


Figure 4.8 The effect of FKBP52 on apoptosis and necrosis in Hela cells. Cells were transfected with the siRFKBP52 FLAG plasmid then left for 48 hrs. Cells were then harvested at 48 hr and stained with Annexin and PI before flow cytometry a) Graph representing the flow cytometry results n=2 b) Flow cytometry data showing percentage of cells live, Apoptotic Early Apoptotic and Necrotic in Hela cells. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns.


Figure 4.9 The effect of FKBP52 on apoptosis and necrosis in U20S cells Cells were transfected with the siRFKBP52 FLAG plasmid then left for 48 hrs. Cells were then harvested at 48 hr and stained with Annexin and PI before flow cytometry a) Graph representing the flow cytometry results n=2 b) Flow cytometry data showing percentage of cells live, Apoptotic Early Apoptotic and Necrotic in U20s cells. Data is graphed as mean +- standard error of the mean from 3 independent experiments. T tests were used to compare siPCMV6-FLAG and PCMV-FLAG FKBP52. ns = > 0.05. data was ns.


4.7 FKBP52 depletion does not affect cell proliferation

In order to investigate the effect of FKBP52 depletion on cell proliferation,

U2OS cells were transfected with control siRNA or FKBP52 siRNA. At 48 hrs post

transfection when it was verified that depletion of the FKBP52 had occurred, cells

were transferred to a 96 well plate and imaged every 24 hrs on an InCell 2200 Imaging

System. Images were then analysed using the ‘Cell Profiler’ analysis software. As

shown in Figure 4.10, depletion of FKBP52 did not affect the cell proliferation rate in

U2OS cells over a 72 hr period. It may be that HeLa would show a difference however

the experiment was not performed due to time restraints.

The affect of FKBP52 on proliferation was tested because the literature presents

FKBP52 depletion or blocking leading in endometriosis to promotion of growth of

endometriotic lessions, with increased cell proliferation inflammation and

angiogenesis (Hirota et al., 2008). Reduced levels of FKBP52 have also been shown

to reduce proliferation of prostate cancer cells but both effects appear to be exclusively

hormone related most likely part of the FKBP52 interaction with the co-chaperone

mechanism and SHR (De Leon et al., 2011).


Figure 4.10 FKBP52 has no effect on cell proliferation. U2OS cells were treated with siControl or siFKBP52. Cells were then seeded and monitored every 24 hr and images were taken to determine cell density. Cell numbers were determined using cell profiler data was then graphed to determine proliferation rate of cells data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare sictrl and siFKBP52. ns = > 0.05. data was ns.


4.8 FKBP52 depletion has no effect on comet tail length

The comet assay was used to determine if up regulation or down regulation of

FKBP52 has any effect on the repair of double strand DNA breaks in a cell. This assay

measures the tail moment of single cells, which increases in size as more DNA breaks

occur. U2OS cells were depleted of FKBP52 using silencer select siRNA. Cells were

treated with 6 Gy of IR to induce DNA breaks and cells were harvested 15 min and 4

hr post IR with an untreated control. This was carried out to establish if FKBP52

depleted cells had more damage after the initial IR treatment and at 4 hrs post treatment

when DNA repair should have occurred. Figure 4.11 a, shows representative images

of the tail moments in all three cell populations before and after IR treatment.

Quantification of the tail to head ratio from at least 100 cells per sample was performed

using Image J. Depletion of FKBP52 was verified by western blotting (Figure 4.11 b).

The data indicates that similar levels of damage occurs in the FKBP52-depleted cells

as compared to the control cells. It also appears both cell populations have reached a

similar tail length at 4 hrs indicating they are undergoing DNA repair at similar rate.


Figure 4.11 Effect of FKBP52 on DNA damage a) Cells depleted of FKBP52 were irradiated with 6 Gy IR and processed for comet assays. Representative images of comet tails are shown for each sample analysed. b) FKBP52 levels in comet assay lysates c) Graph of measured tail moments for siCtrl and siFKBP52, at untreated (unt), IR+ where sample were harvested immediately after IR and 4hr post IR. Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare sictrl and siFKBP521. ns = > 0.05. data was ns.


4.9 FKBP52 depletion reduces the efficiency of homologous recombination

hSSB1 is best described as part of the homologous recombination pathway

(Richard, Cubeddu, et al., 2011a). Although the comet assay did not indicate a DSB

repair defect in FKBP52 depleted cells, homologous recombination only accounts for

repair of 10% of DSBs (Sonoda et al., 2006) and therefore an HR defect may be below

the detection level of the comet assay so to determine if FKBP52 is required for the

repair of double strand DNA breaks by homologous recombination an ‘in cell' reporter

was used (MCF7DRGFP). The HR assay uses an integrated DR-GFP reporter, which

consists of two differentially mutated GFP genes orientated as direct repeats. The up-

stream reporter consists of the recognition site for the rare-cutting I-SceI endonuclease

and the downstream repeat is a 5’ and 3’ truncated GFP fragment. Transient expression

of I-SceI leads to a double strand break (DSB) in the upstream GFP gene. HR is

required to repair the DSB and results in GFP-expressing cells which are quantified

via flow cytometry. hSSB1 is known to be crucial for this pathway and depletion of

hSSB1 has a detrimental effect on HR activity. If FKBP52 and hSSB1 are co-

regulated, FKBP52 depletion could potentially have an effect on the HR pathway. As

shown in Figure 4.12 there is an approximately 15% reduction in the number of GFP

positive cells in FKBP52 depleted cells compared to control cells, indicating that less

HR has occurred in the siFKBP52 population with this being statistically significant

p= 0.036. When compared to the hSSB1 reduction in HR it could be said that FKBP52

was a minor component of HR. However, this discounts the fact that FKBP52 is an

enzyme and that even with our knockdown may be only partially limiting and thus has

a weak phenotype. This will be investigated at a later point.


Figure 4.12 FKBP52 reduces Homologous recombination efficiency. a) flow cytometry of HR assay. Cells were treated with siFKBP52 to deplete FKBP52 levels, then transfected with the ISCE1 plasmid. Cells were left for 24 hours lifted with trypsin then ran through the flow cytometer to detect GFP signal. Blue dots in the gated area indicate GFP expressing cells. b) Graph of normalised Isce1, siCon, siFKBP52 and sihSSB1 flow cytometry data. Data was normalised to GFP transfection Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare siControl and siFKBP521. ns = > 0.05, * = P < 0.01, *** = P < 0.001.


4.10 Regulation of FKBP52 by ATM/ATR kinases in response to IR

ATM, ATR and DNA-PKcs are the three major PI3k-like kinases that are

known to initiate a signalling cascade in response to DNA DSBs. These signalling

cascades involve the phosphorylation of protein substrates that regulate cell cycle

checkpoints, DNA repair and apoptosis. The consensus phosphorylation motif for

these kinases is SQ/TQ sites, where phosphorylation occurs at the serine (S) or

threonine (T) residue (Traven et al. “SQ/TQ cluster domains: concentrated ATM/ATR

kinase phosphorylation site regions in DNA-damage-response proteins”). Analysis of

the FKBP52 polypeptide sequence revealed two adjacent SQ sites (SQSQ; aa 451-454)

and a TQ site (aa 391-392) (See appendix b). In addition, examination of

PhosphoSitePlus (Hornbeck et al. “PhosphoSitePlus, 2014: mutations, PTMs and

recalibrations”), a database of curated mass spectrometry data, indicated that the SQ

451 site had been reported as a DNA damage induced ATM/ATR phosphorylation site

(Lee & Paull, 2005) . Therefore, the role of ATM and the closely related ATR kinases

in the regulation of FKBP52 was investigated.

To determine if the ATM or ATR kinases are involved in the regulation of

FKBP52 levels and/or its response to IR, ATM and ATR were depleted from U2OS

cells using siRNA. 72 hr post siRNA transfection, cells were exposed to 6 Gy IR then

harvested over a 4 hr period. As shown in Figure 4.13, a minor stabilisation of full

length FKBP52 following IR treatment was observed in control siRNA cells, while in

the ATM-depleted cells a lack of stabilisation was observed even though basal levels

of FKBP52 in non-irradiated cells appeared higher. The 44 kDa FKBP52 band was

stabilised in ATM depleted cells, suggesting that the two FKBP52 variants are

regulated in an opposite manner in response to IR and that this regulation may require


the ATM kinase. ATR depletion seemed to predominantly affect the regulation of the

44 kDa FKBP52 protein, as shown by its stabilisation in ATR depleted cells compared

to control cells (Figure 4.14 b). Taken together, these results indicate that the ATM

and ATR kinases may be involved in the regulation of FKBP52 protein in response to

IR.

4.11 FKBP52 SQSQ sites are phosphorylated in response to IR

A common method to investigate potential phosphorylation of SQ/TQ sites is

the use of a commercially available SQ/TQ antibody that only binds the

phosphorylated form of the SQ or TQ motif. To further investigate if FKBP52 is

phosphorylated in response to IR, the FKBP52-FLAG plasmid was transfected into

U2OS cells. Cells were then irradiated and harvested at 1 hr, 2 hr and 4 hr post IR.

Following lysis, FKBP52 was immunoprecipitated using FLAG-M2 Sepharose beads.

Eluted FKBP52-FLAG was separated on an SDS page gel, and western blotting was

performed using the SQ/TQ antibody. As shown in Figure 4.14 c, two SQ/TQ reactive

bands corresponding to full length and 44 kDa FKBP52 were detected at 2 hr and 4 hr

post IR. Interestingly, Hsp90 was also detected in complex with FKBP52 at 2 hr and

4 hr post IR, confirming this previously established interaction. These results suggest

that FKBP52 is phosphorylated at one or more SQ and TQ sites in cells, in response

to IR.


Figure 4.13 ATM and ATR phosporylations effect on FKBP52 a) U2OS cells depleted of ATM using siRNA then irradiated with 6 Gy of IR lysates were run on SDS page gel and transferred to a western blot. The blot was probed using anti-FKBP52 rabbit (Sigma) anti-ATM rabbit (Cell signaling) and anti- B-actin mouse (Abcam). b) U2Os cells depleted of ATR using siRNA then irradiated with 6 Gy of IR lysates were run on SDS page gel and transferred to a western blot. The blot was probed using anti-FKBP52 rabbit (Sigma) anti-ATR rabbit (Cell signaling) and anti- B-actin mouse (Abcam). c) U2Os cells transfected with PCMV6 FLAG-FKBP52 plasmid then exposed to 6 Gy IR cells harvested and lysates treated with magnetic flag beads to collect FLAG-FKBP52. Western blot performed with lysates and blot probed with anti-HSP90 goat (Abcam) SQTQ Phospho-(Ser/Thr) ATM/ATR Substrate Antibody mouse (Cell signaling) and anti-FKBP52 rabbit (Sigma).


Figure 4.14 Knockdown of ATM and ATR and its effect on FKBP52 nuclear levels a) U2Os cells were treated with siRNA to deplete ATM then exposed to 6 Gy IR. Cells were pre-extracted with David’s IF buffer then fixed with 4% PFA at indicated time points. Cells were probed with anti-FKBP52 rabbit (Sigma) and Dapi nuclear stain. b) Nuclear intensity of FKBP52 measured via incell in sicontrol, siATM and siATR populations. Data is graphed as mean +- standard error of mean from 3 independent experiments. T tests were used to compare siCtrl and siATM or siCtrl and siATR. ns = > 0.05. data was ns.

Inte

nsity

of F

KB

P2


Another way of investigating the ATM and ATR regulation of FKBP52 nuclear

levels post IR, is the use of small molecule inhibitors that target ATM and ATR

function. Ku 55933 (Ku 5) is a selective ATM inhibitor, which shows 100-fold

differential selectivity towards ATM kinase activity. This inhibitor specifically

inhibits DNA repair events that are regulated by ATM (Sarkaria et.al 2012). VE-821

(VE 8) is an ATR inhibitor that presents 600-fold selectivity over related kinases ATM

or DNA-PKcs and blocks ATR signalling in cells. Both Ku 5 and VE 8 were chosen

for their specificity and well documented use in DNA damage events. U2OS cells were

treated with 5nM of each Ku 5 and VE 8 or both in combination for 30 mins. Cells

were then irradiated with 6 Gy fixed as previously described and imaged using the

Delta Vision PDV. As can be seen in Figure 4.15 the ATM inhibitor appears to reduce

FKBP52 nuclear levels in response to IR as compared to the DMSO control. ATR

inhibition had a minor impact on FKBP52 localisation while inhibition of ATM and

ATR as expected resulted in a loss of FKBP52 localisation to chromatin post IR

treatment. This data suggests that the ATM kinase is the major effector of FKBP52

cellular localisation post induction of DNA double strand breaks. This supports the

role of FKBP52 in the DNA repair process. It does contradict the siRNA data where

siRNA knockdown of ATM resulted in an increase in chromosome loading (Figure

4.14). It is not unusual however to see such discrepancies as inhibition of activity may

still allow binding to occur but not the phosphorylation event. With knock out, there

is less protein, so no binding occurs and this can open the site to other kinases with

over lapping function.


Figure 4.15 U2OS cells treated with either DMSO negative control, ATM, ATR inhibitor or both ,30min prior to 6gy IR. Cells were pre-extracted with David’s IF buffer then fixed with 4% PFA at indicated time points. Cells were probed with anti-FKBP52 rabbit (Sigma) anti-γH2AX mouse (abcam) and Dapi nuclear stain.


4.12 FKBP52 451 and 453 SQ sites phosphorylation state affects the formation of

the 44kD band

With previous results indicating that the ATM kinase is a regulator of FKBP52

in response to IR, it was crucial to determine if FKBP52 was a direct target of this

kinase. Two potential ATM kinase sites have previously been identified by a

proteomic study (Matsuoka et al., 2007). These are S451 and S453 (Figure 4.16 a). To

explore these further, site directed mutagenesis was used to mutate the sites to generate

S451A, S453A and S451E, S453E mutants. These mutants are known as phospho-

mimics (Glutamic acid mutants) or Phospho-inactive (Alanine mutants) and are

commonly used in the literature to determine the effect of phosphorylation sites. If

either or both of these sites are targets of ATM then by blocking these phosphorylation

events a lack of IR response may be observed. Both sites were mutated to replace the

serine with a phosphomimic Glutamic acid or an Alanine phospho inactive, either

individually or in combination (Figure 4.16 b). When these mutants were over

expressed, it was observed that the phospho-mimic at S451E and the null phospho

mutant at S453A have similar effects and increase the presence of this lower FKBP52

band as observed by western (Figure 4.16 a and 4.16 b). Also, S451A and S453E

appear to have a lesser effect on the production of this second band. This could indicate

some a dual SQSQ mechanism where one site needs to be phosphorylated while the

other needs to be dephosphorylated to achieve to the formation of this second band.


Figure 4.16 the effect of phospho active and phospho null mutants on the double SQ domain in FKBP52 a) Diagram of FKBP52 domains including the 451SQ and 453SQ sites. b) U2OS cells were depleted of FKBP52 using siRNA then transfected with no vector, empty flag vector, PCMV6 FLAG FKBP52 siResistant, PCMV6 FLAG FKBP52 siResistant S451A, PCMV6 FLAG FKBP52 siResistant S451E, PCMV6 FLAG FKBP52 siResistant S453A, PCMV6 FLAG FKBP52 siResistant S453E. samples were harvested and ran on an SDS page gel then transferred to a western blot. Blot was probed with FKBP52 and Flag to determine expression of plasmids. c) U2OS cells were depleted of FKBP52 using siRNA then transfected with no vector, empty flag vector, PCMV6 FLAG FKBP52 siResistant, PCMV6 FLAG FKBP52 siResistant S451A, PCMV6 FLAG FKBP52 siResistant S451E, PCMV6 FLAG FKBP52 siResistant S453A, PCMV6 FLAG FKBP52 siResistant S453E and irradiated at 6 Gy. samples were harvested 4 hr post and untreated controls. Samples then ran on an SDS page gel and transferred to a western blot. Blot was probed for FKBP52 and Flag.


4.13 Discussion

FKBP52 has many roles in the cell including steroid hormone transport, RISC

complex formation, tauopathies and regulation of NF-κB, FKBP52 (Giustiniani et al.,

2015; Jeong et al., 2016; Lagadari et al., 2015; Martinez et al., 2013; Pare et al., 2013;

Storer et al., 2011), however, not much is known about the role of FKBP52 in the DNA

damage pathways. This study identified FKBP52 as being a gene that is co-regulated

with hSSB1 and a potential substrate of the DNA damage responsive kinase ATM.

FKBP52 was identified to not only be regulated in response to IR, but to also regulate

hSSB1 nuclear translocation in response to IR. FKBP52 was found to associate with

HSP90 an interaction that was increased following IR treatment. HSP90 itself is

required for efficient homologous recombination with the loss of HSP90 function

leading to a loss of stability and recruitment of DNA repair proteins including RAD51.

Indeed, HSP90 is currently the centre of drug development programs within the

biotechnology and pharma industries (Shrestha, Bolaender, Patel, & Taldone, 2016).

This study has demonstrated that FKBP52 translocates to the nucleus following

IR treatment and it also may become post-translationally modified as shown by a lower

molecular weight migrating FKBP52, perhaps indicating that it is cleaved by a

protease prior to its translocation to the nucleus. The nature of this lower molecular

weight band is currently being investigated further.

The data presented in this work supports a role for FKBP52 in the regulation of

the DNA damage response pathway. Indeed, FKBP52 localised to small punctate foci

on the chromatin, resembling those of other proteins involved in the homologous

recombination pathway. Although when we examined FKBP52 localisation with the

marker of double strand DNA breaks γH2AX, we were unable to see significant co-


localisation. This could mean that FKBP52 is involved in other aspects of repair of

DSB’s or that it was located at other structures. These structures could, for instance,

be PML bodies that have been shown to have an intrinsic link to homologous

recombination (Yeung et al., 2011). Another potential theory for this increase of

FKBP52 in the nucleus was that it could be transporting proteins from the cytoplasm

to nucleus and vice versa. This role has already been established for FKBP52 in regards

to steroid receptors where its PPIase function is involved in activation of steroid

hormone receptors and their translocation to the nucleus in conjunction with dynactin

motor proteins and the cytoskeleton (Storer et al., 2011). It has been shown that

FKBP52 and its closely related homologue FKBP51 have an antagonistic function in

the transport and retention of NF-κB in the nucleus as well as its transcription

(Erlejman et al., 2014; Lagadari et al., 2015). Interestingly the depletion of FKBP52

resulted in an increase in hSSB1 protein within the cell yet a decrease in the association

of hSSB1 with the chromatin (Figure 3.14). This suggests that hSSB1 may not be

folded correctly. Loss of hSSB1 function has been reported to result in the up-

regulation of the hSSB1 transcript in what appears to be a negative feedback loop (as

seen in the Richard lab). Consistent with this and shown in the next results chapter I

observed an 8-fold increase of hSSB1 mRNA in FKBP52 depleted cells.

To determine if FKBP52 and hSSB1 interacted Co-IPs were performed. These

required extensive optimisation as the heavy IgG chain of the antibodies is the same

size as FKBP52, making it extremely difficult to separate on the gel. To overcome this

the FKBP52 antibody was crosslinked to the dyna beads, this allowed FKBP52 to be

visualised in the Co-IPs on western blots as a distinct band. This Co-IP identified under

the experimental conditions that hSSB1 and FKBP52 may belong to the same protein

complex. However reciprocal experiments did not detect an interaction. This could be


due to the position of the FKBP52 antibody blocking the site to which hSSB1 would

bind or it could be due to the FLAG epitope interfering with a potential binding site.

To further investigate this, full length FKBP52 and hSSB1 proteins were purified and

a direct interaction assay was performed. From this, it was concluded that hSSB1 and

FKBP52 do not directly interact. This does not rule out that they may exist in protein

complexes through indirect interactions but may reflect the need for an unfolded

hSSB1 polypeptide or post-translational modifications to either FKBP52 or hSSB1,

which we are unable to replicate under our experimental conditions. It is also possible

that the interaction is extremely transient.

To determine if any DNA damage response proteins were co-regulated with

FKBP52, FKBP52 was depleted in U2OS cells by siRNA and protein lysates incubated

with antibodies against known DNA damage response proteins, to see if protein levels

or phosphorylation states were altered in the absence of FKBP52. Phosphorylation of

P-Brca1 S1524, ATM 1981, p53 S15 and γH2AX were up regulated in the FKBP52

depleted cells potentially indicating that genomic instability was occurring, again

supporting a role for FKBP52 in DNA damage repair. Interestingly while ATM 1981

was upregulated in FKBP52 knockdown U2OS cells, the actual ATM polypeptide was

decreased. Further, the Rad51 and Banf1 polypeptides also demonstrated down

regulation. This may suggest that FKBP52 is required for stability of these proteins

within the cell. While hSSB1 may have been expected to decrease due to the

connectivity screen indicating that hSSB1 and FKBP52 were upregulated together, it

did not and this is likely due to a negative feedback loop. For instance, a loss of the

hSSB1 polypeptide results in an increase in hSSB1 mRNA expression. It is possible

that a loss of hSSB1 function may also have the same impact setting off a negative

feedback loop. As shown in the next chapter we know hSSB1 transcript increases 8


fold when FKBP52 is lost suggesting that what hSSB1 is in the cell does not function

correctly (may not be folded correctly). In support of this hSSB1, which is the first

protein to localised to double-strand DNA breaks, was not seen to be recruited to break

sites efficiently when FKBP52 was knocked out despite there being more hSSB1

present within the cell.

The data presented in this thesis indicates that FKBP52 is required for normal

genomic stability and DNA repair. To explore if FKBP52 was needed for cell survival

after IR FKBP52 was depleted in cells by esiRNA and then they were treated with

varying doses of IR. Initial experiments in triplicate using the esiRNA showed that

FKBP52 was required for cell survival following IR insult. It is not possible to

determine the off target effects of esiRNA due to the multiple sequences present. To

do this silencer select siRNA was used with and without rescue with siRNA resistant

ectopically expressed FKBP52. Unfortunately, FKBP52 depletion using silencer

select siRNA did not demonstrate a defect in cell survival. This could be due to a

number of reasons. It is possible that the esiRNA was targeting more than FKBP52,

or that the level or rate of FKBP52 depletion was not as great with the silencer select.

Indeed, esiRNA appeared to have around a 10-20% greater knockdown of FKBP52

than the silencer select. Since I was only achieving a 70% knock down potentially that

30% remaining FKBP52 was enough to continue protecting the cell from DNA

damage events. It was also possible knockdown of FKBP52 for longer periods of

time was required to observe the radio sensitivity. As FKBP52 is part of the HSP90

complex, it is likely that it is involved in protein folding by isomerising proteins prior

to entry into the HSP allowing the correct folding to occur. As seen with inhibition of

HSP90 protein levels of target proteins fall over time as the protein is not correctly

folded and is degraded within the cell. It is therefore possible that FKBP52 like HSP90


needs to be depleted for extended periods of time 4-7 days. To counter this CRISPR

was used to create cell lines with a permanent depletion of FKBP52 by introducing

random missense mutations, what was observed was a completely different phenotype

than was produced by the esiRNA. The CRISPR U2OS C4-3 which targeted exon 4

of FKBP52 had the most pronounced effects. In this cell line, I observed that the only

DDR proteins affected were p53 and hSSB1, with complete degradation of p53 and an

overall increase of hSSB1. Potentially this phenotype could be a result of either more

efficient knock down over a much longer time period, or it could just be clonal effects.

I did attempt to restore FKBP52 to the cell line, but this proved quite difficult as the

C4-3 cell line would not produce endogenous levels of FKBP52 using the plasmids

even though the control population would. This was thought to be due to the stressed

nature of this cell line and the C4-3 cell line is potentially diverting its translation

mechanism from a foreign plasmid when it was prioritising its own DDR genes.

The upregulation of ATM, BRCA1 and H2AX phosphorylation discussed earlier

indicated that even unperturbed cells showed an increase in these markers. This

suggested that the genome of the U2OS cells had become unstable. To measure

directly the occurrence of cytotoxic double strand DNA breaks COMET assays were

employed. The neutral COMET assay measures the occurrence of cytotoxic double

strand DNA breaks by electrophoresis of intact genomic DNA. DNA containing

double strand DNA breaks migrates further than the intact chromosomal DNA. The

more breaks that are present, the longer the "tail moment" of the cell meaning more

damage or less repair has occurred. The result obtained for FKBP52 depletion and

attempted rescue were not as predicted. What they suggest is that depleting cell of

FKBP52 reduces the tail moment in response to IR meaning that the cells have fewer

DNA breaks. This unexpected observation may be explained by changes in chromatin


compaction as the more compact chromatin is, the fewer double strand DNA breaks

are caused by IR as the free radicals generated by ionised water cannot get access to

the compacted DNA. Consistent with this, loss of FKBP52 resulted in perturbed repair

kinetics of double strand DNA breaks. Compaction of chromatin impairs the ability

of DNA repair proteins to gain access to the break as well as preventing the

remodelling of the break site to allow invasion of the sister chromatid. Again it would

be interesting to extend the period of knockdown to determine if these phenotypes

changed over time, it would also be interesting to look for epigenetic markers by IF to

determine if the chromatin is indeed more highly compacted in the absence of

FKBP52. FKBP52 is known to associate with the AR and this protein functions to

remodel chromatin around target gene promoters. It is thus possible that other more

general chromatin remodelling proteins are impacted by the loss of FKBP52. The data

presented did have high p values so the data needs to be further repeated using larger

sample numbers.

With hSSB1 being a major component in HR and FKBP52 and hSSB1 having a

potential link, the HR assay was carried out. What the results showed was that FKBP52

might have a minor role in HR with depletion of HR only causing a 15% depletion in

HR compared to the control. This effect is quite small compared to the hSSB1 knock

down which reduced the cells expressing GFP by 70% however given that the FKBP52

knockdown was for 48hrs it is also possible that the targets of FKBP52 had not been

fully impacted at this point or that FKBP52 was not limiting enough.

ATM and ATR have a regulatory role on the DDR pathways and have been

shown to be recruited to DNA damage sites and recruit other DNA damage proteins

with this in mind, experiments were performed where ATM or ATR were inhibited or

depleted, and the effects on FKBP52 were determined. When ATM or ATR were


depleted, it has a minor effect on the protein levels of the second band of FKBP52.

ATM KD reverses the expression pattern of this second band where it is highest at the

1 hr and 2 hr time points. ATR knock down appears to favour peaks at 1 hr and 4 hr

similar to the original wild type blot before. These results could indicate a minor effect

of ATM or ATR on the protein levels of this second FKBP52 band. The small molecule

inhibition of ATM also leads to a major decrease in FKBP52 nuclear localisation as

assessed by IF, potentially indicating that ATM phosphorylation is required for the

transport of FKBP52 to the nucleus or is needed for its cytoplasmic retention.

Confirmation that FKBP52 is phosphorylated at its SQSQ domain via immuno

detection was important, as currently FKBP52 phosphorylation has only been

demonstrated by mass spectrometry approaches. To achieve this, FLAG-FKBP52 was

immunoprecipitated from cells before and after IR and then immunoblotted using an

SQ/TQ phospho antibody. For another layer of control, it would be useful to add the

phospho null mutants, this would act as a negative control and help determine if the

reaction is specific to either or both SQ site. Although technically challenging, faint

bands were observed in some instances that might correspond to phosphorylated

FKBP52. Similar banding was however observed from both irradiated and non-

irradiated samples, suggesting that this signal was not induced by DNA damage.

Finally, the FKBP52 451A/E and 453A/E constructs were generated to test if

disrupting or mimicking phosphorylation might alter FKBP52 expression. The results

presented a change in expression of this second band with 451E and 453A appearing

to have up regulation or stabilisation of the second FKBP52 band. Potentially this

could indicate that FKBP52 is alternatively phosphorylated on each SQ site to cause

the smaller 44 kDa band to form and is why we see phosphorylation at untreated and

post IR at the SQ site. Potentially the mechanism could be that phosphorylation at


S451 results in formation of this second band and phosphorylation at S453 results in

the prevention of this second band. What is left to determine is the purpose of this

second band and if it has a role in any DDR pathways.

To fully understand the role of this phosphorylation event and second band both

bands could be purified then ran through Mass spec to identify the composition of this

second band and in turn help determine its role. Mass spec could also be used in

conjunction with the ATM and ATR inhibitors to determine if phosphorylation is

caused by ATM or ATR and which sites are targeted by ATM or ATR. Finally, it

would be useful to know when FKBP52 is cleaved into the second band and to

determine the fate of this band. It would also be useful to inquire if it stays in the

cytoplasm or does it move into the nucleus, this could be done by adding different tags

on the C and N terminus then performing either IF or sub cell fractionation to

determine where the fragments are after DNA damage.

Chapter 5: Long term depletion of FKBP52 leads to depletion of p53 96

Chapter 5: Long term depletion of FKBP52 leads to depletion of p53

5.1 Introduction

While siRNA is a powerful tool to use in the study of protein function, it does

have a number of drawbacks. Firstly, depletion of the target can be variable, and the

levels of knockdown rarely result in no detectable protein. This can pose a problem

when the focus is the study of an enzyme and it is not possible to reduce levels required

to detect a phenotype. For instance, with siRNA FKBP52 levels can be reduced to

approximately 30% of the levels of control. If FKBP52 levels are still sufficient to

carry out the reaction with 70% knock down, a phenotype would not be obvious.

Additionally, siRNA has a limited time of knock down, providing only a small window

in which to study the effects of the target depletion. Therefore, a strategy utilising

ablation using the CRISPR system was developed to produce a more consistent knock

down over a long-term time frame, this allowed study of the long-term effects of

FKBP52 depletion.

5.2 Validation of CRISPR clones

To investigate whether decreasing the levels of FKBP52 further and for a

longer duration could produce any effects on the DNA damage Response, the CRISPR

Cas9 system was utilised to target start sites at exon 1, exon 4 and exon 7 using the

primers from Table 1. This system introduces mutations to the chosen site, via NHEJ.

Briefly, the Cas9 system cleaves the target site and then the NHEJ system processes

the DNA ends resulting in loss of genetic code (at low frequency). If the site is re-

ligated without loss of code, it can be cleaved again repeatedly until the site is


eventually mutated. The mutation, in turn, can lead to a frame shift disruption in the

formation of the targeted protein. The procedure was performed in the Hela and U2OS

cell lines to produce single cell clones that were then allowed to grow into monoclonal

colonies to produce the CRISPR-generated FKBP52-depleted cell lines. Figure 5.1

presents western blots demonstrating the variability of the clones and was used to

determine which clone would be used for further experimentation. Exon 1 produced

one successful depletion in the Hela cell line with clone C1-4 and one partial depletion

in exon 4 with C4-2. Whole and partial depletions of FKBP52 were observed in exon

1 in U2OS cells and in exon 4, C4-3 is isolated with a depletion of FKBP52. Exon 7

targets produced no successful depletion of FKBP52. When looking at the protein

expression of FKBP52 in the different clones, of particular interest is the different

banding patterns. These bands could indicate alternate start sites that have produced

truncated proteins. These truncated proteins could be similar to the one seen previously

and produce similar results in these cell lines.


Figure 5.1 Crisper Clones targeting exon 1, 4 and 7 a) Hela CRISPRs with controls exon 1 clones C1-3, C1-4, C1-5, C1-6 exon 4 clones C4-1, C4-2 and exon 7 clone C7-3 b) U2OS cells with controls and exon 1 clones C1-1, C1-2, C1-3 and C4-3 some sample performed in duplicate. Blots were probed with sigma FKBP52 to probe FKBP52 levels. c) Densitometry of FKBP52 52 kDa band normalised to Control 1 of Crisper Hela clones. d) Densitometry of FKBP52 52 kDa band normalised to Control 1 of Crisper U2OS clones.

HelaDensiometry normalised to control

Ctrl 1 1.00Ctrl 2 0.96Ctrl 3 0.78C1-3 0.55C1-4 0.55C1-5 0.99C1-6 0.68C4-1 0.87C4-2 0.25C7-3 0.77

U2OSDensiometry

normalised to control

Ctrl 1 1.00Ctrl 1 1.12Ctrl 2 1.19Ctrl 2 1.06Ctrl 3 1.24Ctrl 4 1.27C1-1 0.32C1-2 0.13C1-3 0.83C1-3 0.66C4-3 0.14C4-3 0.05

c) d)


5.3 FKBP52 CRISPR clones show variation in proliferation rate

To determine the effect of FKBP52 depletion in these clones the proliferation

rate of the cells was tested using the Incucyte. This automated microscope images the

cells every 2hrs over a 72hr period. This data is used to calculate the proliferation rate

of the cell lines and to determine if the depletion of FKBP52 had any effect on the

growth of the cells. In the U2OS CRISPR cell lines, it appears that the C4-3 cell line

had the greatest effect with an increase in its proliferation rate as can be seen in Figure

5.2 a. The other cell lines C1-1, C1-2 and C1-4 do not appear to have much effect on

proliferation with C1-1 only having a minor effect. To determine the rate at which

these cell lines "double" Figure 5.2 c) presents the time in hours, it takes for each cell

line to reach 2-fold confluency normalised to the earliest measure time point. Again,

the graph shows little to no variance between the U2OS control cell line and C1-2 and

C1-4 and a 5hr difference between U20S C1-1 and the control. Finally, U2OS C-4-3

presents the greatest difference by reaching 2-fold confluency within 24hrs as opposed

to 46 hrs, showing again the increased proliferation rate of the C4-3 cell line which

has the largest depletion of FKBP52. In the Hela Cell CRISPR cell lines, it appears

that both the C1-4 and C4-2 cell line have an increased proliferation rate when

compared to the control. Although when comparing the initial doubling rate, it appears

that the C4-2 cell line has no initial difference. Meanwhile, the Hela C1-4 cell line

appears to only have a minor difference when compared to the control. The apparent

difference in the cell lines proliferation rates could be due to the level of KD with

U2OS C4-3 and Hela C1-4 showing the greatest level of KD respectively in their cell

lines. It could also be a result of clonal selection and rescue assays would determine

this


Figure 5.2 Proliferation assay of Crisper cell lines. Cells were plated at 3000 cells per well and allowed to grow for 72 hrs. Cells were imaged every 2hrs and confluency was calculated to determine the proliferation rate of each cell line. a) U2OS Crisper cell lines Con, C1-1, C1-2, C1-4 and C4-3 measured over 72hrs. Confluency was normalised to 2hr control to establish base line. b) Hela CRISPR cell lines Con, C1-4 and C4-2 measured over 72 hrs. Confluency was normalised to 2 hr control to establish base line. c) cross section of U2OS CRISPR data focusing on time it takes to reach 2.0 confluency or doubling time. d) cross section of Hela CRISPR data focusing on time it takes to reach 2.0 confluency or doubling time. Experiments were performed in duplicate (n=2)

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5.4 CRISPR clones show a radio-resistant phenotype

Clonogenic assays were performed with the FKBP52 CRSPR cell lines to

investigate if the greater FKBP52 knockdown would potentiate the radio-sensitivity

effects observed using FKBP52 siRNA. However, instead of increase sensitivity, there

was a minor trend in U2OS C1-1, no effect in C1-2; and radio-resistance of C1-4 and

C4-3 compared to the control cell lines (Figure 5.3 a). This is in contrast to the original

siRNA experiments, which presented data that lead to the conclusion that FKBP52

depletion led to radio-sensitivity. The Hela cell line also shows conflicting data with

the C1-4 cell line showing radio-resistance and the C4-2 cell line showing radio-

sensitivity. However, it is important to note that the C4-2 clone appears to have the

highest level of remaining FKBP52 with 43% remaining compared to the controls.

While the cell lines that show resistance to radiation U2OS C1-4 U2OS C4-3 and Hela

C1-4 have 4%, 6% and 16% FKBP52 remaining respectively. These levels are much

lower than anything produced by the either siRNA used in this project and the

difference in results could represent of the threshold of FKBP52 depletion that leads

to a radio-insensitivity. Although unlikely there is the possibility that all the clones

U2OS C1-4 U2OS C4-3 and Hela C1-4 have an off-target deletion that is causing the

different phenotype. This, however, could be checked in the future using a rescue

experiment with wt FKBP52.


Figure 5.3 The effect of FKBP52 depletion on cell survival after DNA damage a) Clonogenic assay of CRISPR cells treated with IR b) western blot of Clonogenic assay cells and probed using anitFKBP52, values under FKBP52 represent remaining FKBP52 normalised to control. Data is graphed as mean +- standard error of mean from 3 independent experiments. T tests were used to compare U2Os control and U2Os CRISPR clones or Hela control and Hela CRISPR clones. ns = > 0.05, *= P = < 0.05.


5.5 Long term depletion of FKBP52 leads to decreased p53 protein levels

Next, the effect of FKBP52 depletion on cell signalling was looked into to

determine if the presence of FKBP52 was required for proper cell signalling function

following induction of DNA damage.

I next chose to examine the C4-3 clone as it had the greatest knock down of

FKBP52. This presented a complete loss of total p53 (Figure 5.4), a protein important

in apoptosis and other roles in DNA damage signalling and repair (Bieging, Mello, &

Attardi, 2014). Although this was the only clone to produce this phenotype, so it is

unknown whether this was due to the efficiency of the FKBP52 depletion as this clone

had the best depletion of FKBP52; or it could be that targeting exon 4 produced this

effect by producing truncated protein, as this clone was the only exon 4 clone identified

with sufficient depletion of FKBP52. Finally, it could be due to some off-target effects

produced by the CRISPR process.

This depletion of p53 could explain the increased IR resistance of the C4-3 cell

line. p53 is required for the apoptosis of cells, and with the C4-3 cell line lacking this

crucial protein in the pathway, it could be possible with a lack of p53 these cells could

not initiate apoptosis and therefore were more likely to survive than the control

population.


Figure 5.4 Effect of Crisper Depletion of FKBP52 on DNA damage proteins: western blot of U2Os con, C4-3. Cells were exposed to 6 Gy IR and harvested at 1,2,4 and 6 hrs post IR. Cells were lysed and proteins harvested and ran on an SDS page gel then transferred to a western blot. Westerns were probed with the above antibodies to determine protein levels.


5.6 FKBP52 depletion causes an increase in p53 and hSSB1 mRNA

To investigate if p53 was being depleted via a decrease in transcription in clone

4-3, RT-PCR was performed to examine the p53 mRNA levels. Primers targeting

FKBP52 at exon junctions 1-2 and exons 4-5 p53 at exon junction 2 and 3 and exon

junction 8 and 9 and hSSB1 were utilised for RT-PCR (Figure 5.5). These experiments

confirmed, as anticipated that FKBP52 RNA levels were reduced by the CRISPR

process compared to the control cells. Also of great interest was the increase of the

p53 transcript in the C4-3 cells. This indicates that the depletion of FKBP52 does not

reduce the level of p53 transcription but causes an increase in p53 transcription of up

to 5-fold. This suggests that p53 is being reduced at the protein level and not at the

transcript level, indicating that p53 is likely being degraded as a result of low FKBP52

levels. An example of this is with hTERT, where FKBP52 is required for the transport

of hTERT to the nucleus and if the hTERT is not transported to the nucleus it is

degraded in the cytoplasm (Jeong et al., 2016; Lagadari et al., 2016).

hSSB1 transcript levels were also examined and appear to show a 6-fold

increase in the C4-3 population of cells. This again could indicate that FKBP52 is

required for normal hSSB1 function and without functional hSSB1 protein the

feedback loop is activated and more transcript made.

5.7 Inhibition of the proteasome via MG132 restores p53 protein levels

Next, the protease inhibitor MG132 was used to determine if protein

degradation was causing the loss of p53. MG132 is a compound, which reduces the

degradation of ubiquitin-conjugated proteins in mammalian cells, inhibiting the

proteasome and in turn preventing the degradation of proteins turned over by the

proteasome. MG132 has been shown to activate c-Jun N-terminal kinase (JNK1),

which in turn initiates apoptosis. MG132 also inhibits NF-κB activation with

https://en.wikipedia.org/wiki/Ubiquitin

https://en.wikipedia.org/wiki/C-Jun_N-terminal_kinase

https://en.wikipedia.org/wiki/Apoptosis

https://en.wikipedia.org/wiki/NF-%CE%BAB


an IC50 of 3 µM and prevents β-secretase cleavage. When this inhibitor was used in

the C4-3 cell line, it leads to an increase in p53 levels indicating that FKBP52 absence

leads to the proteasomal degradation of p53.

In regular proliferating cells, p53 is maintained at a low concentration and

occurs predominately in its inactive form with a small half-life of 15-30 minutes. This

is generally maintained by an interaction between p53 and MDM2 (Mouse Double

Minute 2 homolog) the main negative regulator of p53 (Shi & Gu, 2012). Activation

of p53 via post translational modification such as phosphorylation at Ser 15 and 37

leads to a dramatic and rapid increase in p53 levels and prevents the degradation of

p53 via MDM2 ubiquitination. Phosphorylation at Ser 15 and 37 also increase p53

affinity for p300, in turn stimulating acetylation of p53 via p300 (Meek & Anderson,

2009). Acetylation of p53 via p300 leads to activation of sequence specific DNA

binding of p53. This acetylation also leads to the stability of p53, leading to higher

levels of p53 in the cell.

With this in mind, the levels of p300 and HDAC1 were measured using

immunofluorescence. This data shows that in the C4-3 cell line that p300 levels are

down and that HDAC1 levels appear to be up (Figure 5.7 and 5.8). This indicates

potentially that an upstream regulator of p300 could be causing the downstream effect

of p300 degradation. As can be seen in Figure 5.6 addition of the MG132 inhibitor

appears to not only recover p53 levels as previously observed but also appears to

increase p300 levels and slightly increases H3K56 acetylation, again indicating a

potential effect of FKBP52 knockdown on not only the p300-MDM2-p53 pathway but

on acetylation in general.

https://en.wikipedia.org/wiki/IC50

https://en.wikipedia.org/wiki/Beta-secretase_1


Figure 5.5 q-RTPCR of U2OS control and U2OS C4-3 CRISPR cells. Cells were exposed to 6gy IR and harvested 1,2,4 and 6 hours post IR. Cells were lysed and RNA was harvested and q-RTPCR was performed with primer pairs targeting FKBP52, hSSB1 and p53 exon junction 2-3 and 8-9. Data is graphed as mean +- standard error of mean from 2 independent experiments. T tests were used to compare U2Os Control and C4-3. ns = > 0.05, *= P = < 0.05, ** = P < 0.01, *** = P < 0.001.

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Figure 5.6 Effect of MG132 on P53 and H3 acetylation. U2OS control and C4-3 CRISPR cells. Cell were exposed to DMSO, Mg132 8hrs. cells were then harvested and lysed in RIPA buffer proteins harvested and ran on an SDS page gel then transferred to a western blot. westerns were probed for p300, MDM2, p53 D07, FKBP52, actin, H3 L56 acetylation and H4.


Figure 5.7 the effect of FKBP52 depletion on nuclear p300 and HDAC1.U2OS Control and U2OS C4-3 CRISPR were fixed with 4% PFA.Cells were probed for FKBP52 HDAC1, p300 and Dapi to stain the nucleus. a) Images of U2OS control CRIPSR and C4-3. incell analysis of the cells measuring the intensity of fluorescence in the CRISPR cell lines U20s con, C1-1, C1-2,C1-4 C4-3, Hela Con, Hela C1-4 and Hela C4-2. b) FKBP52 intensity c) p300 intensity, d) HDAC1 intensity.


a)

b)


Figure 5.8 the effect of MG132 on the protein levels of p300 and H3K56Ac in FKBP52 depleted cells. U2OS Control and U2OS C4-3 CRISPR were treated with DMSO (control) or MG132 25um for 16hrs then fixed with 4% PFA.Cells were probed for p300 and H3 Lysine 56 acetylation.a) incell anaylsis of the cells measuring the intensity of fluorescence detecting p300. b) incell analysis of the cells measuring the intensity of fluorescence of H3 Lysine 56 acetylation.


5.8 FKBP52 complementation to restore p53 phenotype

To determine if the p53 levels could be rescued when FKBP52 levels were

restored, C4-3 cells were transfected with Flag-FKBP52 plasmid. After 24 hrs cells

were harvested lysed and western blotting was performed. Initial experiments did not

increase FKBP52 levels to endogenous levels in clone 4-3 making it difficult to

determine if p53 restoration could occur with FKBP52 recovery (Figure 5.9). This

experiment was attempted again but the plasmid was allowed to express for 72 hrs.

This time point was chosen with the theory that longer exposure to the plasmid would

produce higher levels of FKBP52 for a longer time and potentially stop the degradation

of p53.

The transfection appeared to produce high levels of FKBP52 in the CRISPR

control Vector leading to higher than endogenous levels but the same procedure failed

to increase FKBP52 to endogenous levels in the C4-3 cell line. This recovery

experiment had no effect on p53 levels although the results are still inconclusive as the

level of FKBP52 did could not reach endogenous levels in the C4-3 clone. The C4-3

cells have elevated levels of p53 and hSSB1 transcript, this upregulation of proteins

involved in the DDR could be the reason why the FKBP52 plasmid was not producing

as much protein when compared to the U2OS control. With the cell expending the

majority of its energy on transcribing DNA repair proteins.


Figure 5.9 Attempted rescue of FKBP52 in the C4-3 cell line. U2OS control and C4-3 CRISPR cells were transfected with Flag-FKBP52. cells were transfected and after 24hrs cells were exposed to 6 Gy IR then harvested at 1,2 and 4 hrs post IR.


5.9 Inducible hyperacetylation restores p53 levels

With previous experiments showing that transcription of p53 is increased in

the C4-3 cell line and that MG132 a proteasomal inhibitor restores p53 levels, it was

hypothesised that acetylation or reduced levels of acetylation might be responsible for

the degradation of p53. Using Sodium butyrate (NaBU) to induce hyper acetylation

over a 16hr period it was predicted that p53 levels would be restored.

As can be seen from Figure 5.10 and Figure 5.11 NaBU caused hyper

acetylation of H3 L56 indicating the treatment worked. Also of interest is the levels of

endogenous H3 acetylation appear slightly lower in the C4-3 cells than the U2OS

control cells potentially indicating that FKBP52 depletion could have an effect on

Histone Deacetylases (HDAC) or Histone Acetyl transferases (HATS). When looking

at the p53 levels, it can be seen that NaBU induced hyper acetylation has resulted in a

down regulation of p53. This leads to the prediction that FKBP52 degradation has an

effect on an upstream regulator of the acetylation of p53. This, in turn, would prevent

the activation of p53 and also lead to the degradation of p53. It is perplexing that in

the C4-3 cell line the two regulators of p53, p300 and MDM2 also appear to be low or

non-existent until NaBU is added. Possibly MDM2 levels could be low due to its levels

being regulated by p53 protein levels in the nucleus as they form a negative feedback

loop with p53 levels regulating MDM2 levels. Or the reduction of FKBP52 could be

effecting an upstream regulator of the p300-MDM2-p53 mechanism.


Figure 5.10 Effect of Sodium butyrate (NaBU) on P53 and H3 acetylation. U2OS control and C4-3 CRISPR cells. Cell were exposed to PBS (con) or NaBU for 8hrs. cells were then harvested and lysed in RIPA buffer proteins harvested and ran on an SDS page gel then transferred to a western blot. westerns were probed for p300, MDM2, p53 D07, FKBP52, actin, H3 L56 acetylation and H4.


Figure 5.11 the effect of MG132 on the protein levels of p300 and H3K56Ac in FKBP52 depleted cells. Control and U2OS C4-3 CRISPR were treated with PBS (control) or NaBU 10 mM for 16hrs then fixed with 4% PFA. Cells were probed for p300 and H3 Lysine 56 acetylation. a) incell analysis of the cells measuring the intensity of fluorescence detecting p300. b) Incell analysis of the cells measuring the intensity of fluorescence of H3 Lysine 56 acetylation.

a)

b)


5.10 Discussion

The CRISPR Cas9 system is a new tool in which a gene can be permanently

edited, in this case, to be "switched off". The most popular CRISPER-Cas9 system

consists of three key components (Cong & Zhang, 2015): the endonuclease Cas9,

CRISPR RNA (crRNA, and transactivating crRNA (TracrRNA). Additionally, the

tracrRNA-crRNA duplex can be fused to form a chimeric sgRNA for efficient use in

in gene editing. The sgRNA consist of a 20 nucleotide guide sequence complementary

to the target site. This sgRNA binds to the target sequence via Watson-Crick base-

pairing and guides Cas9 to accurately cleave the DNA strand, forming a double strand

break at the site. These sites are processed by NHEJ which leads to random

insertion/deletion mutations in the sequence which ideally lead to a frame shift

mutation, which in turn inactivates the gene.

This system was chosen to look at FKBP52 depletion due to previous siRNA

depletion systems not producing consistent results and not reducing FKBP52 to

desired levels. It was thought that by using the CRISPR system I would be able to

determine if any of the effects I saw with siRNA were real and consistent. What was

observed was interesting, the initial creation of a 100-90% knockdown was quite

difficult and only occurred in two cell lines of the initial 15 produced. The U2OS C4-

3 cell line and the Hela C1-4 cell line, the remaining cell lines either had no effect from

the CRISPR or produced a variation of banding patterns which could be FKBP52 from

alternate start codons that were unaffected by this process. Some of these may mimic

the band that was mentioned earlier in this thesis but due to time constraints I was

unable to investigate this. When I performed the Clonogenic survival assay on the

acceptable CRISPER cell lines, I observed results contradictory to the siRNA

experiments. With U2OS C1-4, C4-3 and Hela C1-4 now showing a radio resistance


with depleted FKBP52 levels as opposed to the radio sensitivity or no effect that the

siRNA experiments produced. This could be due to a threshold that FKBP52 levels

need to reach for this radio resistance to be reached. Although another theory as to why

the U2OS C4-3 cell line survived was that this cell line had lost its p53, a protein

required for apoptosis to occur. When looking at proteins involved in DNA damage

pathways, the C4-3 cell line again shows data different to a siRNA knockdown.

Previously KD of FKBP52 presented an increase in P-P53, P-ATM, P-BRCA1, P-

yH2AX. Now the data is showing a complete reduction in p53 and upregulated levels

of hSSB1 (although this was consistent with the siRNA). Although this is a fascinating

phenotype, it has only occurred in the one U2OS crisper clone that demonstrated the

lowest levels of FKBP52. This presents the question, is this an effect of FKBP52

depletion or is this an effect of off target CRISPR effects. One other theory is that

previously the experiments only produced a max 70-80% KD over a three-day period

and that a greater knockdown for a longer duration was required to achieve this

phenotype. To try and determine if FKBP52 depletion was the cause of the p53

depletion a rescue was attempted using the previously designed FKBP52-HA-FLAG

plasmid. Unfortunately, I was unable to reach basal levels with this plasmid in the C4-

3 clone even when doubling the concentration and expression time of the plasmid. This

could be due to the cells being so stressed and damaged that they do not favour the

transcription of the plasmid over their own repair and maintenance. Evidence for this

is that the same plasmid transfected into the control cells at the same time and same

procedure produced a much better expression. Although I was unable to restore the

FKBP52 and p53 levels in these cells, I continued to establish why p53 levels were

depleted. Using quantitative PCR, it can be seen that the levels of both p53 and hSSB1

have been drastically increased. This is more than likely due to the cells initiating a


feedback loop system. To compensate for a loss of p53 and functional hSSB1, the cells

increase the transcription of these genes. The increase of hSSB1 also indicates that loss

of FKBP52 does potentially lead to genomic instability and the up-regulation of hSSB1

transcript levels to compensate. The hSSB1 response was also observed in the U2OS

C1-2 cell line which still had partial FKBP52 remaining and no p53 defect. The use of

the MG132 inhibitor provided evidence that the depletion of FKBP52 was post-

translational and due to proteasomal degradation, this lead to the investigation of the

p300-MDM2-p53 mechanism. Results showed that MDM2 and p300 were also

reduced in the C4-3 cell line, indicating that a potential upstream regulator of this

mechanism could also be affected by FKBP52 depletion. The use of NaBU to increase

p53 and to a lesser extent MDM2 and p300 lead me to believe that potentially an

HDAC or HAT are being effected (Bagrel et. al, 2004) which is resulting in a reduction

in acetylation of the chromatin and certain sites and potentially p300 as well. However,

we observe more p53 transcript levels when FKBP52 is depleted suggesting that there

is no transcriptional reason for the lack of p53 but more likely a proteasomal response.

The further increase in p53 levels following NaBU treatment may be in response to

even higher rates of p53 transcription. IF data does show a reduction in acetylation at

H3Lysine56 in the C4-3 cell line as well as an increase in HDAC1 that targets this site

and could potentially explain this defect. HDAC1 is usually upregulated after DNA

damage (Jackson et. al, 2010). Overall the CRISPR data presents some interesting

results. Although some of these results differ from the siRNA, this may all be

explained by FKBP52 only being partially depleted in the siRNA treated cells while

more extensively depleted in the CRISPR knockouts.

Chapter 6: FKBP52: a potential survival indicator in lung cancer 120

Chapter 6: FKBP52: a potential survival indicator in lung cancer

6.1 Introduction

Targeting the DNA damage response pathways in cancer cells is recognised

as a therapeutic option in a number of cancers. Indeed, most of the first line

chemotherapeutics function by causing genomic damage within cancer cells. With

evidence that FKBP52 could be regulated by ATM/ATR and has a role in the DDR,

it was sought to determine if FKBP52 was a viable cancer target. This involved

Kaplan-Meier data analysis to determine if there was any association between

FKBP52 expression and the patient outcome in lung, gastric and breast cancers.

When it was found that FKBP52 expression can predict the outcomes in Lung cancer

and Breast cancer patients this was investigated further.

6.2 Kaplan-Meier data indicates that FKBP52 up or down regulation can be an

indicator of survival in cancer

Kaplan-Meier plotter was used to establish if FKBP52 mRNA expression has

prognostic potential in cancer. It was found that high levels of FKBP52 were

associated with poor prognosis and reduced survival in a number of cancers such as

non-small cell lung cancer adenocarcinoma (Figure 6.1) and Estrogen Receptor

positive (ER positive) and Progesterone Receptor positive (PR positive) breast cancer.

Inversely, low expression correlated to a poor outcome in gastric cancer (Figure 6.2).

The role of FKBP52 in the ER and PR positive cancers was as expected and likely due

to the reported role of FKBP52 as a co-chaperone of Hormone receptors. Just as the

ER negative and PR negative cancer types were unaffected by FKBP52 levels of


mRNA expression, as the cancers no longer required the steroid hormone receptors to

survive meaning FKBP52 function no longer effected the health of these cells.


Figure 6.1 FKBP52 in lung and gastric cancer. Kaplan-Meier plots of FKBP52 expression levels and survival of patients with lung cancer, gastric cancer, squamous small cell lung cancer and adenocarcinoma. Plots were retrieved from kmplot.com.


Figure 6.2 FKBP52 in breast cancer. Kaplan-Meier plots of FKBP52 expression levels and survival of patients with breast cancer, breast cancer, was split into estrogen positive estrogen negative progesterone positive and progesterone negative. Kaplan-Meier plots were retrieved from kmplot.com.


6.3 FKBP52 protein expression in a lung cancer panel

To explore the role of FKBP52 in breast and lung cancer further, a panel of

available cell lines for lung and breast cancer were used. Results showed that in lung

cancer cell lines when compared to normal immortalised epithelial cell lines (HBEC),

FKBP52 protein levels appear to be low with the exception of the HCC827 cell line

(Figure 6.3). This cell line has an activating mutation within the tyrosine kinase

catalytic domain of the epidermal growth factor receptor (EGFR), which causes an

upregulation in EGFR activity (Zhong et al., 2007). EGFR is a tyrosine kinase and has

been shown to target and phosphorylate FKBP52 at tyrosine residues. Phosphorylation

of FKBP52 by EGFR results in inhibition of adeno-associated virus 2 (AAV2) second

strand synthesis (Zhong et al., 2007) however, the mechanism underlying this is not

clear. It is possible that the constitutively active EGFR could explain why the FKBP52

polypeptide is up regulated in this cancer cell line compared to the other lung cancer

cell lines tested. This would be an interesting avenue to explore in the future.

6.4 FKBP52 protein expression in a breast cancer panel

Analysis of the breast cancer cell line panel, demonstrated as expected, that the

FKBP52 levels are higher in ER positive cell lines when compared to ER negative cell

lines (Figure 6.4). This result is consistent with the literature as in breast cancer cell

lines where ER is upregulated (e.g. MCF7) FKBP52 levels also appear to increase

indicating that FKBP52 is upregulated in hormone dependant cancers. FKBP52 itself

does not bind to ER but instead the immunophilin cyclophilin 40 (cyp40) (Ratajczak

& Ward, 2015) . These results also show that FKBP52 expression differs in a cancer

context and indeed if FKBP52 is to be a therapeutic target in cancer then FKBP52

levels may function as a companion diagnostic.


Figure 6.3 Expression of FKBP52 in a lung cancer cell line panel. a) Western blots of FKBP52 and Hsp90 in a panel of lung cancer derived cell lines: HCC827 adenocarcinoma with an EGFR mutation, H2228 adenocarcinoma non-small lung cancer, H460 large cell lung carcinoma, and SKMES squamous cell carcinoma and normal immortalised epithelial cells HBEC3, HBEC4, HBEC5, β-actin was used as loading control. b) Densitometry data of HSP90 and FKBP52 in lung cancer derived cell lines normalized to HBEC3.

a) a)

b)


Figure 6.4 Expression of FKBP52 in a breast cancer cell line panel. a) Western blot of FKBP52 and HSP90 in breast cancer derived cell lines consisting or the ER positive cell lines: MCF7, T47D, MDA175, MDA361, MDA486, and the ER negative cell lines: MDA468, BT549, BT20 SUM159. The cell lines PMC42ERT and PMC42LA were also included. Western blot was probed for HSP90α, FKBP52 and β-actin. b) Densitometry data of HSP90 and FKBP52 in Breast cancer cell panel normalised to MCF7.

a)

b)


6.5 TMA data indicates that over or under expression of FKBP52 could indicate

poor prognosis

To determine if there was any correlation between FKBP52 protein levels in

lung cancer and patient outcome, a tissue microarray was stained for FKBP52. This

TMA staining allowed me to compare this with the KM plot data to determine if there

was a correlation between protein expression levels and patient outcomes. Results

were split by nuclear and cytoplasmic staining to determine if there was an association

between the subcellular location of FKBP52 and patient outcome. Looking at the

overall combined data of the adenocarcinoma and the squamous cell carcinoma shows

that there is a non-statistical trend of poor survival in cells with higher FKBP52 levels

in the nucleus, when compared to the cytoplasmic expression (Figure 6.5 & 6.6).

Cytoplasmic FKBP52 levels showed no difference in survival outcome between high

and low FKBP52 staining (Figure 6.6). When the populations are split by cancer type,

the adenocarcinoma population presented a trend towards high FKBP52 expression

both in the nucleus and cytoplasm, associating with better survival. This data contrasts

with the squamous population of patients, which only shows a different survival

outcome in the nuclear staining, with high nuclear staining associated with poorer

survival outcome. Of particular interest is the stark contrast of the KM plotter Data

compared to the TMA data presented here. Where high FKBP52 expression leads to

lower survival outcomes in adenocarcinoma cancer patients in the KM data, but in the

TMA data set the lower level of FKBP52 protein expression lead to poorer survival

out comes. This inversion of outcomes occurred again in the squamous population

where squamous presented better survival outcomes in correlation with


Figure 6.5 TMA of FKBP52 in lung cancer patient samples. Nuclear staining blue line indicates tumours with a score lower than 150, and the red line indicates tumours with a score greater than 150 with a min of 0 and a max of 300. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only c) squamous count only.

Survival of FKBP52 Nuclear All dichotomized: Survival proportions


Figure 6.6 TMA of FKBP52 in lung cancer patient samples. Cytoplasmic staining. Blue line indicates tumours with a score lower than 150, and the red line indicates tumours with a score greater than 150 with a min of 0 and a max of 300. a) combined adenocarcinoma and Squamous count. b) adenocarcinoma count only c) squamous count only

Survival of FKBP52 Cytoplasmic All dichotomized: Survival proportions


upregulated FKBP52 mRNA, but reduced outcomes when protein was upregulated in

the TMA.

The data was next separated by the score, to determine if the intensity of FKBP52

staining influenced survival. Cells were scored as follows; 3+ (strong): staining visible

at low level of magnification (x4 objective lens), 2+ (moderate): staining visible at

intermediate level of magnification (x10 or x20 objective lens) 1+ (weak): staining

only reliably confirmable at high level of magnification (x40 objective lens) 0

(negative): no staining visible at a high level of magnification.

From this data an interesting pattern emerged, it appeared that patients with a

score of 0 indicating no FKBP52 staining or patients with a score of 3 indicating high

intensity FKBP52 staining, had reduced survival outcomes. When separating into the

two cancer types, this effect was best represented in squamous cancer cells and further

stratifying of the data showed nuclear staining best represented this effect. It was

interesting that mid-range scores of 1 and 2 had better outcomes, showing that FKBP52

has an optimal range where it improves patient survival. This could be due to FKBP52

being a critical regulatory protein, were if it is over or under expressed it disrupts

downstream functions required for patient survival. It is also possible that expression

is linked to specific genetic or phenotypic backgrounds. Our TMA data did not have

associated genetic data, so we were unable to determine if other genetic traits were

associated with FKBP4 expression in the 0 and 3 score groups.


Figure 6.7 TMA of FKBP52 in lung cancer patient samples. Nuclear staining scores have been separated into 0, 1, 2 and 3. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only c) squamous count only. Log-Rank tests were used to compare 0, 1, 2 and 3. ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Survival of FKBP52 Nuclear 0-3 All: Survival proportions


Figure 6.8 TMA of FKBP52 in lung cancer patient samples. Cytoplasmic staining scores have been separated into 0, 1, 2 and 3. a) combined adenocarcinoma and squamous count. b) adenocarcinoma count only c) squamous count only. T tests were used to compare 0, 1, 2 and 3. ns = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Survival of FKBP52 Cytoplasmic 0-3 All: Survival proportions


6.6 Discussion

FKBP52 has previously been shown to have a role in hormonal cancers

involving steroid receptors, in particular, prostate cancer and the AR receptor. This

current data indicates that FKBP52 also has differential expression in breast cancer,

with this appearing to be linked to the oestrogen receptor (ER) status, this is consistent

with its role as a steroid hormone receptor transporter, with FKBP52 levels being lower

in the ER negative cell lines.

I also examined a lung cancer TMA. It can be seen that FKBP52 expression

varies depending on the type of lung cancer, with KM plots showing that in

Adenocarcinoma, high expression of FKBP52 mRNA was associated with a poor

survival outcome. However, compared the survival analysis of patients stratified by

FKBP52 protein levels, it is apparent that there is an opposite survival outcome, where

high protein levels of FKBP52 are associated with better outcome. Again, this

inversion of patient outcome also occurs in the squamous cell carcinoma population

where highly expressed FKBP52 mRNA is associated with a better prognosis while

low protein levels of FKB52 lead to a better prognosis. The data was next stratified to

score four different expression levels of FKBP52. Interestingly this revealed that when

FKBP52 levels were very low (0 score) or when they were very high (3 score) that the

patients presented a lower probability of survival when compared to low and moderate

level expression (1 & 2 representative scores). This potentially indicates that there are

two populations of lung cancer cells, perhaps with distinct genetic backgrounds that

are associated with poor outcomes. One where FKBP52 promotes growth perhaps due

to enhanced repair of DNA damage and a further cell line where low FKBP52 allows

growth through perhaps the inhibition of apoptosis. It would be worth looking at


genomic instability levels and apoptosis in lung cancer cells in the future to determine

if these correlate with FKBP5 protein expression.

Chapter 7: Discussion 135

Chapter 7: Discussion

My PhD thesis has three distinct but connected aims as outlined below.

1. To investigate the interaction between hSSB1 and FKBP52 in vivo and in

vitro

2. To investigate the role of FKBP52 in response to IR-induced DNA damage.

3. Investigate if FKBP52 has a role in cancer

My hypothesis was that FKBP52 plays a critical role in the DNA damage

response pathway, likely through its enzymatic role as a proline isomerase.

7.1 FKBP52 and hSSB1

My project looked at the potential role of FKBP52 in the DNA damage

response, as preliminary data had indicated that the DNA repair protein hSSB1 and

FKBP52 were co-regulated (connectivity screen performed by the CARP Laboratory

at QUT). My initial theory was that FKBP52 would interact with hSSB1 via the co-

chaperone mechanism to modify and transport hSSB1 to the nucleus, as this is the

canonical role that FKBP52 performs in the cell, such as with the GR receptor and

other hormone receptors (Storer et al., 2011). FKBP52 has also been shown to be

required for the transport of other DNA damage proteins such as NFKB, p53 and

hTERT and to be involved in their stability and/or transcription (Galigniana, Harrell,

O'Hagen, Ljungman, & Pratt, 2004b; Jeong et al., 2016; Lagadari et al., 2015).

Additionally, hSSB1 contains a proline rich domain that may be a target for FKBP52.

FKBP52 depletion however, appeared to have the opposite effect to what was

predicted by the connectivity screen. Depletion of FKBP52 led to an 8-fold increase

in hSSB1 mRNA levels and an increase in hSSB1 protein levels was also observed.

This indicated that the co-regulation of hSSB1 and FKBP52 might not be as simple as


first thought, and it raised the possibility that without FKBP52, hSSB1 activity was

lost and the cell responded by inducing mRNA expression of hSSB1. A previous study

of hSSB1 indicated that hSSB1 functions with IntS3- a member of the integrator

complex, to regulate the transcription of the hSSB1 gene (Skaar et al., 2009) and that

hSSB1 is on a negative feedback loop whereby loss of hSSB1 protein levels results in

an increase in mRNA expression. If FKBP52 is required for the correct folding of

hSSB1 then in cells lacking FKBP52, hSSB1 may not be correctly folded, non-

functional and thus triggering the feed-back loop. This is further supported by

immunofluorescence data showing that hSSB1 is not being correctly transported to the

nucleus so is unable to function in the DNA repair processes in the absence of

FKBP52. It is also possible that FKBP52 depletion produces downstream effects that

leads to DNA damage or lack of repair, which results in an activation of the ATM

kinase and an increase of hSSB1 transcription and protein stability. The strongest

phenotype was observed in the FKBP52-CRISPR depleted cells where almost all

FKBP52 was depleted, while a similar but milder phenotype was observed in FKBP52

siRNA depleted cells where approximately 70% of FKBP52 was lost. These results

did indicate that FKBP52 may have an important role to play in the DNA damage

response pathways through the regulation of hSSB1 activity.

Next, I explored further the possibility that FKBP52 interacted with hSSB1.

To investigate this initially co-IPs were performed, these experiments are especially

difficult due to FKBP52 being roughly the same size as the IgG heavy chain, with IgG

masking preventing the detection of FKBP52. By using anti-hSSB1 antibody

conjugated to protein G Dynabeads I was able to pull out FKBP52 at 1 hr and 2 hrs

post IR indicating that hSSB1 and FKBP52 formed a complex in response to IR-

induced DNA damage. Unfortunately, I was unable to perform the reciprocal


immunoprecipitation experiment, which could be due to steric hindrance resulting

from the FKBP52 antibody binding to the same domain required for the hSSB1

interaction.

Further investigation of the antibody revealed it had been raised against a

peptide that included the FKBP52 FK1 domain, its active PPIase domain, the THR

hinge domain (which is required to be phosphorylated to activate FKBP52) and the 2nd

inactive FK domain. This indicates that it is plausible that since the antibody has

antigenicity against up to three domains of the protein, two of which have been shown

to be required for its function (Bracher et al., 2013), that steric hindrance could be

involved. To resolve this, it may be possible to use either an antibody that recognises

another part of FKBP52 or to use ectopically expressed FKBP52 that has an N or C

terminal FLAG-tag. It would be possible that an interaction could be detected using a

FLAG-FKBP52 to immunoprecipitate the protein without the risk (or with reduced

risk) of steric hindrance occurring. Of course, as well as the antibody potentially

inhibiting the interaction of hSSB1 with FKBP52 it is also possible that the antibody

could not bind to FKBP52 that was already bound to hSSB1. It has been shown that

inhibition of the FK1 domain or competitive inhibition using over expressed FK1

domain can inhibit the co-chaperone mechanism.

FLAG-FKBP52 was attempted to determine the interaction with hSSB1

however, this did not produce a reliable result and due to time constraints, this was not

able to be further investigated. Instead, direct interaction between hSSB1 and FKBP52

was performed. The proteins were purified in a bacterial system and recombinant His-

tagged hSSB1 was bound to cyanogen bromide beads and added to recombinant His-

tagged FKBP52. This experiment determined that under the conditions used in this

study that FKBP52 and hSSB1 do not directly interact, although there were limitations


to this experiment. FKBP52 was purified in a bacterial system where post translational

modifications do not occur such as phosphorylation of Serine residues. If the

interaction was dependent on a phosphorylation event by the DNA damage responsive

kinases ATM, ATR and DNA-PKcs then a direct interaction in my conditions would

not be observed. Potentially without such post translational modification of FKBP52,

it would not be in a favourable confirmation to bind to hSSB1.

Another theory is that FKBP52 is in complex with hSSB1 and does not directly

interact with it but requires other elements of the co-chaperone mechanism such as

HSP90 or p23. These proteins have been shown previous to form complexes with

FKBP52 co-chaperone targets and usually requires HSP90 binding to the target protein

e.g. a GR then FKBP52 would bind to HSP90 via its TPR domain before interaction

with the target protein (Davies et al., 2005; J. Li, Soroka, & Buchner, 2012; Riggs et

al., 2003). To investigate this possibility, the experiment could be repeated where

HSP90 and p23 could be included in the appropriate ratios to determine if FKBP52

requires them to bind to hSSB1. Also, it would be interesting to modify FKBP52 at

known and putative ATM/ATR/DNA-Pkcs phosphorylation sites (SQSQ) to

determine if these need to be phosphorylated before FKBP52 can interact with hSSB1

directly.

What can be concluded about hSSB1 is that FKBP52 depletion appears to

upregulate mRNA and protein levels of hSSB1. My data also shows that FKBP52 and

hSSB1 may interact in vivo and that when FKBP52 is depleted, hSSB1 levels increase

in the cell, however, hSSB1 which is normally a nuclear protein appears in the

cytoplasm (see Figure 3.2). This leads to the current theory that FKBP52 is required

for the correct folding and translocation of hSSB1 to the nucleus in response to IR.


7.2 The role of FKBP52 in response to IR-induced DNA damage.

In this thesis, I also investigated the response of FKBP52 to IR treatment. IR

produces both single and double strand DNA breaks. While single stranded DNA

breaks are repaired by the base excision repair pathway relatively simply, the double

strand DNA breaks pose a larger problem. While hSSB1 has been shown to be

involved in the repair of double strand DNA breaks it is not yet known if it also plays

a role in the repair of single strand DNA breaks. The results from the connectivity

map, as well as the new data generated by this thesis, implies that FKBP52 may be

regulated by similar mechanisms to hSSB1. My results indicate that FKBP52 does

respond to IR treatment with an initial depletion of FKBP52 protein levels and the

appearance of a smaller novel 44 kDa band, that was a suspected cleavage product of

FKBP52. Over time the FKBP52 levels recovered. Interestingly, however,

immunofluorescence analysis of the FKBP52 response demonstrated that following IR

FKBP52 localised to distinct nuclear foci reminiscent of those seen for hSSB1.

Significant fluctuations in the transcriptional levels of FKBP52 were not observed

indicating that IR response was likely due to changes in the post translational

regulation of FKBP52 or a partner protein.

To understand if FKBP52 levels were regulated by cell cycle and potential

checkpoints FKBP52 levels throughout the cell cycle were also measured. Cells were

synchronised using a double thymidine block, which arrests cells in the G1/S phase.

FKBP52 levels appeared to be highest in the G1 phase of the cell cycle. Interestingly

while this may imply a role in NHEJ and not HR I observed that loss of FKBP52

resulted in a defect in HR activity. This of course would be consistent with the loss of

hSSB1 activity within the cell.


My data indicated that FKBP52 has a critical role in the regulation of the DNA

repair protein hSSB1. hSSB1 as well as being required for HR is also absolutely

necessary to initiate cellular checkpoints. To determine the effects on the DNA damage

response in vitro in control and FKBP52 depleted cells (siRNA and the CRISPR Cas9)

I looked at survival of depleted cells, proliferation and the effect on cell signalling.

Initially, results suggested that FKBP52 depletion using siRNA lead to an increase in

the phosphorylation of DDR proteins such as P-BRCA1, P-ATM, P-p53, P-CHK2,

yH2AX and an increase in basal levels of hSSB1, all this indicates that FKBP52

depletion leads to cells undergoing increased DNA damage repair. This in conjunction

with the clonogenic assays which showed radio sensitivity of FKBP52 depleted cell

and a loss in the ability to repair breaks by HR, presented a story of a protein involved

in the DDR. However, FKBP52 depletion appeared to have no effect on the comet tail

length, an indicator of DNA double strand breaks. This indicates that depletion of

FKBP52 does not affect the repair of DNA double strand breaks which is contradictive

with the HR assay. It is possible however that a defect in FKBP52 results in all double

strand DNA break repair occurring through NHEJ. This could explain the observed

lack of comet tails in the FKBP52 depleted cells. While the depletion of FKBP52 by

siRNA showed increased signalling the CRISPR data revealed that FKBP52 depletion

did not induce signalling but may suppress signalling, a phenotype that would be

consistent with a loss of hSSB1 function within these cells. Indeed, while there were

difficulties in getting the ATM 1981 antibody functioning, the data did suggest that

signalling to BRCA1 was impaired and that p53 signalling and protein was depleted.

This is particularly interesting as MDM2, a protein known to target p53 to the

proteasome was also depleted in the FKBP52 CRISPR knockout. These discrepancies

between CRSIPR and siRNA are very likely due to differences in how complete the


loss of FKBP52 is within the cells. For instance, while siRNA gave a 70% knockdown

the CRISPR gave a nearly complete loss of the FKBP52 protein (>95%), as determined

by western. In this case, if the impact was due to the hSSB1 loss, we do see that a

partial loss of hSSB1 results in genomic instability with enhanced signalling, however,

a >90% loss of hSSB1 sees HR being abrogated and a loss of cellular signalling. If

FKBP52 is not completely limiting in the case of the siRNA, then it is likely that

hSSB1 would be only partially depleted and thus being sufficiently present to cause

signalling. Clonogenic survival assays also support this concept as partial depletion of

FKBP52 leads to radio sensitivity, while CRISPR deletion resulted in resistance likely

due to a loss of p53 protein.

Possible future experiments could be performed to look at the capacity to

rescue cells depleted of FKBP52 by ectopic over-expression of FKBP52. It should also

be considered if further FKBP52 CRISPR clones which sufficient loss of FKBP52

could be used to confirm my observations. Or it could be possible that FKBP52 has

multiple roles and these are confounding the experimental results. For instance, radio

resistance could be coming from the loss of p53 function in the FKBP52 depleted cells.

This would mean even if double strand DNA breaks were occurring the cells would be

protected from apoptosis. It is also possible that loss of FKBP52 forces the cell to use

NHEJ which is a more rapid method for break repair which may thus provide

protection for normally lethal doses of radiation.

The CRISPR C4-3 cell line data indicated that complete KD of FKBP52

reduced p53 levels. This was contradictory to the previously published observation

that showed depletion of FKBP52 had no effect on endogenous p53 but increased did

increase p-p53. This is likely to be explained by our own data that indicates the levels

of FKBP52 are important. The potential reason that p53 levels are diminished could


be that with no FKBP52 present, p53 remains in the cytoplasm to be poly ubiquitinated

by MDM2. However, I also observed a loss of MDM2 in the C4-3 cell line. This could

be explained by the low levels of p53 (a transcription factor) as MDM2 and p53 are in

a negative feedback loop where up regulated p53 levels lead to upregulation of MDM2

transcription, and p53 decrease leads to an MDM2 decrease (Nag, Qin, Srivenugopal,

Wang, & Zhang, 2013). Interestingly p53 transcription is quite high in FKBP52

knocked out cells. P300 is a protein involved in the regulation of p53 through its HAT

domain and acetylation via p300 regulates p53 and MDM2 activity and p300 through

auto acetylation. P300 polypeptide is also low in FKBP52 depleted cells indicating

that if the machinery that regulates p53 stability has been reduced then potentially,

FKBP52 depletion has led to epigenetic changes through upstream regulators such as

an HDAC or HAT. This mechanism requires further investigation to discover the

effects of FKBP52 on acetylation.

From this study, I have found that FKBP52 protein levels and cellular

localisation changes following induction of double strand DNA breaks by IR. Partial

depletion of FKBP52 results in enhanced signalling while near complete loss of

FKBP52 results in suppression of p53, MDM2 (protein levels) and BRCA

phosphorylation. What must be considered is that p53 and MDM2 may not be folding

correctly and thus their half-life is reduced. It would be helpful to look at the unfolded

protein response in FKBP52 depleted cells. Consistent with this, both MDM2 and p53

loss can be rescued with the proteasome inhibitor MG132.

7.3 Is FKBP52 regulated by ATM or ATR phosphorylation?

A proteomic study looking for ATM/ATR substrates identified two IR induced

phosphorylation sites on FKBP52 located at consensus sequences S451 and S452


(Matsuoka et al., 2007). This SQ domain is a motif known to be targeted by the ATM,

ATR or DNA-PKcs Kinases, which are known to respond to DNA damage stimulus

and trigger the DNA damage response. Initial experiments tried to determine the effect

that ATM and ATR have on FKBP52 using siRNA and the KU inhibitors. These

experiments indicated that the novel FKBP52 44kDa band was differently regulated

by the reduction of ATM or ATR kinases, indicating that this band may have a role in

the DDR response. Through IPs, I also presented data that showed that FKBP52 is

phosphorylated at an SQ/TQ site in response to IR consistent with the previous study.

Interestingly siATM appeared to increase FKBP52 levels in the nucleus (indicated via

IR) where inhibiting ATM using Ku 55933 seemed to reduce FKBP52 nuclear levels.

This event was possibly due to inhibition activity maybe allowing binding to occur but

not the phosphorylation event, meaning that the site could not be phosphorylated by

other kinases. It is possible that ATR and or DNAPK could also have over lapping

substrate affinity and thus where no ATM protein was present ATR or DNAPK could

be phosphorylating the site. This could be tested in the future by performing the

FLAG-FKBP52 IP in cells using the inhibitors and siRNA then probing with the

phosphor SQ/TQ antibody. This would indicate if the above theory is true. I also tried

to confirm the SQ phosphorylation sites using null mutants for S451A and S453A that

cannot be phosphorylated. Unfortunately, even with optimisation, I was unable to get

this experiment to work. My mutants failed to express to equivalent levels, and the

commercial P-SQ/TQ antibody sometimes failed to detect even the positive controls.

Potentially other antibodies could be optimised and attempted, or fresh mutants with

different tags could be attempted for future experiments to determine if 451SQ or

453SQ are phosphorylated in response to IR. It would be interesting to determine if

ATM or ATR CO-IP with FKBP52 in response to IR. Potentially a similar method that


was used to determine the in vivo interaction between FKBP52 and hSSB1 could be

attempted in the future to determine if these two proteins interact but these proteins

would need to be expressed in a eukaryote organism to be sure that the appropriate

post-translational modifications occur. It would also be of interest to perform mass

spectrophotometry in conjunction with an IP to map phosphorylation events on

FKBP52 following IR.

Finally, to determine the effect of phosphorylation at the double SQ domain, I

created phospho mimics (S451E/S453E) and phospho null mutants (S451A/S453A) at

these sites. A previous study had identified S451 and S453 as potential ATM, ATR

phosphosites and that these events were induced by IR. ATM, ATR or DNA-PKcs, are

known regulator of the DNA damage response and all have the same SQ/TQ consensus

sequence. The phospho mutants indicated that the phosphorylation state of S451 and

S453 determined whether the second FKBP52 band would be present. It appeared that

phosphorylation at S453 (the S453E mutant) allowed this second band to form and

lack of phosphorylation at S453 (S453A mutant) inhibited this protein. This indicates

that the formation of this second band is regulated by either ATM or ATR and that

both sites act as a switch to allow for the cleavage of FKBP52 into its lower band.

What remains to be determined are the composition of this second band and the effects

of this cleavage event. I believe future studies, FKBP52 mutants could be solely

expressed in the CRISPR FKBP52 depleted cells, with no competition from

endogenous FKBP52. This could allow for the determination of the effect of this

phosphorylation event. Essentially by repeating the survival, cell sensitivity and cell

signalling assays but with cells solely expressing the S451A, S451E, S453A, S453E

mutants, the profile of phosphorylation at the SQ sites could be achieved. It would

also be useful to extract both the endogenous and lower band in a mutant and wild type


context and use mass spectrophotometry or either N or C terminal sequencing to

determine the site of cleavage. This would also aid in determining its function. Finally,

it would also be insightful to determine if this second band is localised to the cytoplasm

or the nucleus, this could be performed via sub cell fractionation and probing using the

FLAG antibody. Once a cleavage site is determined a mutant of this site could be used

to determine the impact this event can have on the function of FKBP52.

7.4 FKBP52 and lung cancer

TMA data was used to the explore the role that FKBP52 may have in a real

cancer context. Previous studies have shown that FKBP52 may have a role in hormone

dependent cancer such as prostate cancer, which is due to the role that FKBP52 plays

in the transport of Steroid hormone receptors. Until this thesis, a link between lung

cancer and FKBP52 had not been established. With the realisation that dysregulation

of FKBP52 can indicate a poor prognosis in Adenocarcinoma and squamous cell

carcinoma, this can lead to a new avenue of research to attempt to determine if

FKBP52 has a role in lung cancer and if known therapeutics that target FKBP52 and

the co-chaperone mechanism could be used in the treatment of this disease.

7.5 Model of FKBP52 function in DNA damage response

Based on findings of this thesis it is possible that FKBP52 is phosphorylated at

451S in response to IR and dephosphorylated at 453S to form this second band. What

needs to be determined is how the protein is cleaved and the consequence of this

cleavage event. The data presented in this thesis for the first time describes FKBP52

as a critical protein functioning in the DNA damage response and apoptosis pathways.

FKBP52 appears to regulate the function of hSSB1, p53 and MDM2 within the cell.


My work has also shed light on how FKBP52 is regulated within the cell in response

to IR. The data demonstrates that FKBP52 is regulated both by phosphorylation and

an apparent protease driven cleavage of FKBP52. The function of this cleavage is yet

unknown but raises the possibility that it performs DNA damage specific functions

within the cell. Lastly, my thesis explored the role FKBP52 may play in cancer. It

highlights that either low or very high levels of FKBP52 are associated with poor

prognosis, and this would be in part supported by my experimental work. Cells low

in FKBP52 would be predicted to be deficient in apoptosis induced by p53 while

gaining genomic instability required for cell evolution into cancer. Cells expressing

high FKBP52 may on the other hand, have high levels of homologous recombination

meaning they may show resistance to DNA damaging chemotherapeutics. Those

patients expressing intermediate levels of FKBP52 similar to our siRNA knockdowns

have enhanced rates of apoptosis and genomic instability making them susceptible to

chemotherapeutics that cause DNA damage.

Figure 7.1 represents the proposed model devised from the findings of this

thesis. I propose that in response to a DNA damage event such as IR ATM, ATR or

DKA-PKcs dephosphorylates FKBP52 at S453 and phosphorylates FKBP52 at s451

(Figures 4.13 c 4.16 a and b). This triggers a cleavage event by an unknown protease

and creates the 44 kDa FKBP52 fragment (Figure 4.1). Next either full length or

potentially the cleaved FKBP52 protein in conjunction with the HSP90 co-chaperone

mechanism transport hSSB1 to the nucleus leading to an accumulation of hSSB1 in

the nucleus (Figure 3.2 a and 3.4 a and b). This then allows for DNA damage to be

repaired by mechanisms such as HR (Figure 4.12).


Overall, it can be concluded that FKBP52 is an important protein in the regulation of

the cell response to DNA damage. The novel findings presented in this thesis will aid

as a standing point for future more detailed studies to help elucidate the different roles

of FKBP52 in the DNA damage response. It has now been demonstrated that FKBP52

plays more crucial role than initially thought in the DDR pathway and requires

regulation by ATM/ATR to fulfil its role. It has also been shown in this thesis that

FKBP52 protein levels can indicate the likelihood of survival in lung cancer patients,

which is novel for FKBP52 and I believe future directions for this project would

involve looking into the role FKBP52 may play in lung cancer.


Figure 7.1 Proposed model for FKBP52 response to IR. DNA damage event occurs which in turn leads to the phosphorylation of FKBP52 at S451 and dephoshorylation at S453. This leads to recruitment of hSSB1 and the HSP90 co-chaperone mechanism which in turn transports hSSB1 to the nucleus.

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Appendices 160

Appendices

Appendix A

Validation of siRNA, antibodies and plasmids.

Two commercially available antibodies were tested to detect FKBP52 by

western blotting: anti-FKBP52 (Sigma-Aldrich Prestige) was raised in rabbit, and anti-

FKBP52 Hi52C (Abcam) was raised in mouse. As can be seen in Figure (A1.1.) the

epitope for the Sigma antibody targets the two conserved PPiase domains. These

domains do share homology and conserved sequences with FKBP51. This target

sequence for the polyclonal antibody covers a 64 amino acid stretch with 46.87%

identical residues; this could potentially lead to false readings of FKBP52 as FKBP51.

The epitope for the Abcam antibody has not been mapped by Abcam, so could not be

included in this thesis. The specificity of the two antibodies were tested via lysates

depleted of FKBP52 using siRNA. As shown in Figure A1.2 both antibodies

specifically detect the FKBP52 protein at the predicted molecular weight of 52 kDa.

However, the mouse anti-FKBP52 Hi52C had a stronger affinity for a non-specific

band than for FKBP52 and required a 1/500 dilution, while the rabbit anti-FKBP52

(Sigma-Aldrich Prestige) predominantly detected FKBP52 and required a 1/5000

dilution to detect FKBP52 in protein lysates. It was thus decided to use the anti-

FKBP52 Sigma Prestige antibody for western blotting. Also of interest is the effect of

the siRNA on the proteins in this blot. silencer select was utilised for the antibody

optimisation experiment, and it can be seen that the larger band detected has not been

affected by the siRNA, indicating it is a nonspecific product. However, the lower band

detected by the Sigma prestige antibody appears to be depleted by the siRNA indicating

it could be another form of FKBP52, such as a cleaved form.

Appendices 161

Throughout the project, two types of siRNA against FKBP52 were used:

esiRNA (Sigma-Aldrich) and silencer select (Thermo Fisher). The esiRNA is a

heterogeneous mixture of 21 bp oligos covering a large section of the target gene

sequence. This method was the initial method chosen as it had been previously shown

in the lab to produce effective and persistent depletion of FKBP52. However, as

esiRNA contain a pool of different sequences they cannot be used in rescue

experiments, which require expressed constructs to be resistant to depletion with a

single sequence of siRNA. For this reason, a sequence-defined siRNA sequence was

also chosen.

Appendices 162

Figure A1.1 Comparative protein sequence of FKBP51 and FKBP52. The epitope target sequence (in yellow) of the Sigma Aldrich Prestige FKBP52 antibody. Sequence covers area with 46.87% similarity.

Appendices 163

Figure A1.2 Comparison of FKBP52 antibodies. Cells were treated with siRNA targeting FKBP52 or a scramble control (siCtrl), protein was extracted and ran on a 12-4% SDS page gel and transferred to a nitrocellulose membrane. The first blot is the Sigma antibody only in grey scale. The second blot is the Abcam antibody only in grey scale.

Appendices 164

The defined sequence FKBP52-targeted siRNA was in the form of “silencer

select” from Thermo-Fisher, which is a locked nucleic acid chemistry that is thermo

stable and has a greater affinity to the target site than standard siRNA technologies

(Elm n, 2005). silencer select was also used due to its successful use to target other

proteins of interest within the CARP laboratory at QUT. Both types of siRNA types

were shown to be efficient at decreasing the FKBP52 protein levels up to 96 hr post

transfection (Figure A1.3 a and b). However, at the maximum recommended

concentration of 10 nM, silencer select appears to knock down FKBP52 protein faster

and more efficiently than esiRNA (Figure A1.3 b). To further optimise the silencer

select siRNA, concentrations of 5 nM and 10 nM were tested with transfection reagent

RNAimax at 1:1 and 1:2 ratio to siRNA, i.e. 1 pmol of siRNA: 0.1 µl RNAimax is 1:1

ratio and 1 pmol siRNA to 0.2 pl RNAimax is 1:2 ratio (Figure A1.3 c and A1.3 d). It

was concluded that the optimal conditions to achieve maximal knock down were 10

nM siRNA and a 1:2 ratio of siRNA: RNAimax (e.g. 2.5 µl siRNA: 5 µl RNImax with

the oligo at a concentration of 40nM). A slightly better effect was observed in the U2OS

cell line compared to Hela cells (Figure A1.3 e and A1.3 f). Figure A1.4 illustrates the

FKBP52 cDNA sequence, indicating the Sigma-Aldrich esiRNA sequence coverage

(green) and the silencer select target region (blue). As can be seen, esiRNA covers a

larger sequence of the FKBP52 cDNA than the silencer select. Due to the esiRNA being

composed of thousands of different sequences, with each at very low concentrations, it

has been demonstrated that there are very low off target effects from this technology.

Both types of siRNA were used throughout this project as indicated.

Appendices 165

Figure A1.3 Depletion of FKBP52 using esiRNA and silencer select siRNA. a) western blots of U2OS cells transfected with esiRNA targeting FKBP52 or an esiRNA targeting the green fluorescence protein GFP as a control. Cells were harvested at 48,72 and 96 hr post transfection. c)-f) western blots of Hela and U2OS cells transfected with silencer select siRNA at 1:1 or 2:1 ratio of RNAimax: siRNA, and harvested at 48,72 and 96 hr post transfection. All westerns were probed using antiFKBP52 rabbit (Sigma) and anti-β-actin mouse (Abcam).

Appendices 166

Figure A1.4 FKBP52 human cDNA sequence. Green highlighted bases indicate the esiRNA target sequence, the blue highlighted sequence shows the silencer select target sequence.

Appendices 167

Next, the two antibodies against FKBP52 were tested for use in

immunofluorescence (IF) microscopy. U2OS cells transfected with FKBP52 or control

siRNA were fixed and probed with the Sigma-Aldrich FKBP52 antibody (Figure A1.5).

It was observed that FKBP52 localised in the cytoplasm and the nucleus of non-

extracted cells (Figure A1.5 a). This was consistent with previous results that showed

FKBP52 to occur throughout the cell (Sanchez, 1990). To investigate whether FKBP52

localised to the chromatin, cells were treated with an extraction buffer to remove all

soluble proteins (as used previously Richard et al., 2008 (Nature). In pre-extracted cells,

the levels of FKBP52 were reduced in both the cytoplasm and nucleus (Figure A1.5 b),

suggesting that a proportion of FKBP52 exists in a soluble form in both the nucleus and

the cytoplasm of the cell, and a proportion of FKBP52 is bound to the cytoskeleton and

chromatin within the cell. The use of siRNA validated the specificity of the FKBP52

antibody in immunofluorescence as shown by greatly reduced staining levels in both

the pre-extracted and non-extracted FKBP52 depleted cells. The Hi52C monoclonal

antibody was also tested and was found to produce too much background signal to be

useful for IF procedures.

Appendices 168

Figure A1.5 Immunofluorescence staining of FKBP52 a) Immunofluorescence of U2OS cells that have been treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were fixed using 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus.b) Immunofluorescence of U2OS cells that have been treated with a non-specific siRNA (siCtrl) or and siRNA targeting FKBP52 (siFKBP52). Cells were pre-extracted to remove -soluble cellular proteins prior to fixation in 4% PFA. Cells were probed with anti-FKBP52 antibodies (Sigma) and DAPI was used to stain the nucleus.

Appendices 169

It is important to show the effects of siRNA-mediated depletion of a protein are

specific and to do this, a siRNA resistant transcript of the gene of interest is expressed

in "rescue" experiments. To provide this tool to validate the siRNA experiments, a

siRNA resistant FKBP52 plasmid was generated in the PCMV6 vector. To confirm the

siRNA resistance of the plasmid, U2OS cells were first transfected with siRNA

targeting FKBP52 at 10 nM, followed by transfection with the empty Flag plasmid and

the siRNA-resistant Flag-FKBP52 plasmid at 48 hrs post siRNA transfection. As

shown in Figure A1.6, expression of the siRNA-resistant FKBP52-Flag polypeptide

was readily detectable confirming that the cDNA was indeed resistant to the siRNA.

Also of note is that the ectopically expressed FKBP52 also expressed a lower migrating

band at approximately 44 kDa that was detected by both the FKBP52 antibody and the

Flag antibody. This indicates that FKBP52 may be processed by cleavage events, a

topic that is further addressed later in this thesis (Chapter 3.2, 4).

Appendices 170

Figure A1.6 Expression of PCMV-6-FLAG-FKBP52 siRNA resistant plasmid. Cells were treated with siRNA FKBP52, then (-) empty PCMV6FLAG vector or a PCMV-6-FLAG-FKBP52 siRNA resistant vector were transfected. Protein lysates were run on a 4-12% SDS-Page gel and transferred to a western blot then probed with anti FKBP52, Anti-Flag and anti β-actin antibodies.

Appendices 171

Appendix B

FKBP52 structure and function

With FKBP52 overexpression correlating with poor prognosis in cancer patients

and differential expression in cancer cell lines, the physical structure of FKBP52 was

analysed from data available through a literature search and websites such as

Phosphosite (http://www.phosphosite.org/) and Uniprot (http://www.uniprot.org/). It

has already been established that FKBP52 consists of four domains: a PPIase domain,

a PPIase-like domain that binds nucleotides but has no PPIase function, the TPR

domain, a tandem repeated sequence of 34 amino acids through which it binds HSP90,

and the Calmodulin binding domain (described in detail in Chapter 1). DNA repair

kinases such as ATM, ATR and DNA-PKcs regulate DNA damage signalling and

usually phosphorylate SQ/TQ motifs (Matsuoka et al., 2007). Examining the FKBP52

sequence, it was observed that FKBP52 also contains two SQ sites that could potentially

be phosphorylated. These potential sites were discovered through mass spectrometry

screening procedures and are predicted to be phosphorylated by ATM kinase at SQ451

and by MAD2BP kinase at SQ453 (Matsuoka et al., 2007) MAD2BP is a protein

involved in mitosis and cell cycle regulation (Ma, Chan, Chen, On, & Poon, 2012).

As previously established in the literature FKBP52 has a homologue, FKBP51

(Storer et al., 2011). This protein shares 70% homology with FKBP52 and contains a

potential SQ phosphorylation site at Serine 445. Both proteins are believed to have

evolved from FKBP12, which only contains the PPIase domain (Figure B.1 a)

(Heitman, Movva, & Hall, 1991; Jun Liu et al., 1991). FKBP52 and FKBP51 have been

described to have antagonistic functions to each other, which may be explained in part

through their very similar 3D structures (Figure B1.1 b). FKBP52 is highly conserved

Appendices 172

in mammals except at the C terminus where the potential SQ phosphorylation sites are

located. The high level of conservation between the FKBP52 and FKBP51 polypeptides

in mammals suggests the protein has a critical function and that it evolved before the

divergence of the mammals (Figure B1.2).

Appendices 173

Figure B1.1 Comparative structure of the FKBP family a) Comparative domain map showing the similarities between FKBP12, FKBP51 and FKBP52. All three proteins contain the PPIase domain. FKBP51 and FKBP52 also contain a PPIase-like domain that binds to ATP and GTP and a TPR domain that binds HSP90. The Calmodulin binding domain is only present in FKBP52. b) FKBP51 and FKBP52 X-ray crystallographic structures. Figures adapted From Storer et al 2011

Appendices 174

Figure B1.2 Amino acid comparison of the human FKBP52 and its homologous protein FKBP51. Highlighted sections indicate domains: yellow is the PPIase domain, green is the PPIase-like domain and purple is the TPR repeat domain. Full lines indicate a direct match, : indicates a conserved residue, . indicates a semi conserved residue and a blank space indicates no conservation.

Appendices 175

Figure B1.3 Amino acid sequence comparison of FKBP52 in human, mouse, rat and rabbit. Red amino acids are small amino acids, blue are acidic amino acids, magenta are basic amino acids, green are the hydroxyl, sulfhydryl amine and grey indicates unusual amino acids. Asterisk (*) indicate positions which have a single, fully conserved residue. A colon (:)indicates conservation between groups of strongly similar properties. Period (.) indicates conservation between groups of weakly similar properties.

Appendices 176

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