supporting information - pnas · 2020. 5. 29. · [35s]methionine/cysteine (easytag™ express35s...
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SUPPORTING INFORMATION
MATERIALS AND METHODS
Reproducibility
All experiments were performed a minimum of two times; where repeated more often, it is so
indicated in the figure legend.
Cell Lines and Culture
HeLa, U2OS, HCT116 and HEK293T cells were grown in DMEM media with 10% FBS at 370C
and 7.5% CO2. Cell lines were tested for mycoplasma contamination. Drosophila SF9 cells were
grown at 270C in TNM-FH insect medium (BD Biosciences Pharmingen).
Plasmid constructs.
Murine 6X His-BRD4-Flag WT and ∆N, ∆C, ∆B1B2 and ∆HAT mutants are as described
previously (1). The 6X His-BRD4Δkinase-Flag mutant was generated as a drop-out mutant
through PCR using the BRD4 WT construct in the pACHLTc insect vector as the template. The
murine WT BRD4 and BRD4 Δkinase mutant were moved into the mammalian pCDNA3.1 vector
by excising the BRD4 sequences with BamH1 and SmaI and subcloning them into pCDNA3.1
using BamH1 and EcoRV. The SmaI and EcoRV sites were destroyed in the process. The plasmids
bearing murine WT c-Myc and c-Myc T58A mutant in the pcDNA DEST40 mammalian
expression vector was a gift from Rosalie Sears (Addgene plasmids 45597 and 45598). MYC WT,
1-369aa, 1-270aa, 1-63aa, 63-270aa, 63-163aa and 144-270aa mutants were cloned into the pGEX
bacterial expression vector to be expressed with a GST tag. The pBV-Luc wt MBS1-4 Luciferase
reporter construct with CDK4 core promoter E-boxes was a gift from Bert Vogelstein (Addgene
plasmid 16564). The CMV-HA epitope tagged ubiquitin expression plasmid (pMT123) was a gift
from Allan Weissman (National Cancer Institute)
Reagents
Antibodies used for either immunoprecipitation or immunoblotting were Anti BRD4 (H-250: sc-
48772) anti-ERK1 (G8: sc-271269) Anti-His probe (H-3: sc-8036) and anti-GST (B-14: sc-138)
from Santa Cruz Biotech, anti-MYC (Y69: ab32072, (2, 3), anti-MYC pThr58 (ab185655) anti-
MYC pSer62 (ab185656) anti-Ac Histone H3K122 (ab33309) and anti-beta tubulin (ab6046) from
Abcam, anti-Ac Histone H3K4 (Millipore), anti-Ac Histone H3 (Upstate), anti-Histone H3 and
www.pnas.org/cgi/doi/10.1073/pnas.1919507117
anti-Histone H4 (Cell Signaling), and Anti-Ubiquitin-biotin (ebioP4D; ThermoFisher Scientific).
Proteosomal inhibitor MG132 (Sigma) and translation inhibitor Cyclohexamide (Sigma) were
dissolved in dimethyl sulfoxide (DMSO) or ethanol respectively to make stock solutions. GSK3β
inhibitors, CHIR-99021 and LY2090314 were purchased from Selleckchem and dissolved in
DMSO to make stock solutions. Stock solutions of inhibitors were stored at -200C.
Purification of Recombinant proteins.
Flag-tagged BRD4 and BRD4 mutants were purified as described earlier (1). Flag-tagged TAF7
was purified similarly. GST-tagged MYC and MYC mutants were expressed and purified from
E.coli cells (BL21 strain, Novagen) by inducing expression using 0.7mM IPTG overnight at 180C
and repeated pull-downs and concentration using GST Sepharose beads (GE Life Sciences). All
purified proteins were concentrated using microcon size exclusion columns (Millipore), recovered
in HKEG buffer (20mM Hepes, pH 7.9, 100mM KCl, 0.2mM EDTA, 20% vol/vol Glycerol) and
stored frozen at -800C. Highly purified (> 99%) human histones H3 and H4 substrates were
obtained from New England Biolabs as recombinant proteins expressed in E.coli; their purity was
confirmed by the company through Mass Spectrometry Analysis (ESI-TOF MS) and peptide
sequencing. Purified recombinant human His-MYC (>95% purity), expressed in E.coli, was
purchased from GeneMed Synthesis. Recombinant p300, expressed and purified from Sf9, with
purity confirmed at > 95%, was purchased from ProteinOne. Purified recombinant kinase active
human untagged ERK1 and His-tagged GSK3β was purchased from Sigma Aldrich.
Transient transfections
Transient transfections of the mammalian expression plasmid constructs containing Myc,
MycT58A, BRD4, BRD4Δkinase or the E-box-Luciferase reporter detailed above were done using
Lipofectamine (Invitrogen) and harvested 18 hrs post transfection except were mentioned
otherwise. Under these conditions, cell viability and growth were not affected by the transfections.
When testing for histone acetylation in vivo, cells were treated with 5 mM Sodium butyrate during
transfection to inhibit HDAC activity. Transfected cells were treated with 10 µM of MG132
proteosomal inhibitor for 4 hrs before harvesting where mentioned in the fig legends. Whole cell
extracts were made from the cells and analyzed by immunoblotting using specific antibodies as
indicated in the figure legends and equal amounts loaded on gels based on total protein quantities.
Co-Immunoprecipitations and in vitro binding assays
To co-immunoprecipitate MYC or ERK1 with BRD4 from HeLa nuclear extracts, magnetic beads
(Dynabeads Protein A) were coated with 5μg of Anti-BRD4 antibody and incubated with 25 μg
HeLa nuclear extract (Promega) for 3hr at 40C. The beads were then washed thrice with 50mM
Tris (pH 8.0), 200 mM NaCl, and 0.2% NP-40. Bound proteins were separated on SDS PAGE gels
and immunoblotted with antibodies against BRD4, MYC or ERK1. For In vitro binding assays,
BRD4 or MYC proteins were pre-incubated for 1hr with either M2 Flag-agarose (Sigma), Ni-NTA
agarose (Qiagen), MYC-agarose (Sigma) or GST-Sepharose (GE Life Sciences) beads and then
incubated overnight at 40C with the appropriate interacting protein mentioned in the figure legend.
The beads were washed twice with 50mM Tris (pH 8.0), 150 mM NaCl, and 0.2% NP-40 and
immunoblotted with the appropriate antibody. All immunoblot analyses were performed using
secondary antibodies from Li-Cor and the OdysseyTM infrared scanner. Specific antibodies as
indicated in the figures were used in these analyses.
Sequential Co-Immunoprecipitations
Nuclei was extracted from HeLa cells using the REAP protocol as described previously(4). Nuclei
were lysed by resuspending in 10mM HEPES pH 7.9, 20% glycerol, 0.2M NaCl, 1.5mM MgCl2,
0.4 mM EDTA, 0.5% NP40, 1mM DTT supplemented with protease inhibitors for 15 mins at 40C.
The lysate was separated into chromatin bound (pellet) and non-chromatin fractions (supernatant)
by centrifuging at 12000g for 10 min at 40C and the pellet solubilized with 10 units of
Benzonase® Nuclease (Sigma-Aldrich) To co-immunoprecipitate ERK1 and BRD4 from
chromatin bound extracts, magnetic beads (Dynabeads Protein A) were coated with 5μg of specific
antibody as described in the figure legends and incubated with 500 μg of the extract for 3hr at 40C.
The beads were then washed thrice with 50mM Tris (pH 8.0), 200 mM NaCl, and 0.2% NP-40.
40% of the immunoprecipitated material was saved for western blots and the remaining 60%
subjected to a secondary immunoprecipitation as described in the figure legends. Bound proteins
were separated on SDS PAGE gels and immunoblotted with antibodies against BRD4, MYC or
ERK1.
Pulse-chase experiments.
HeLa or MEF cells with MZ1 treatment or Cre-viral infections respectively, as indicated in the
figure legends, were plated at a density of 1.6 x 106 cells per 10 cm-diameter dish the day prior to
pulse-chase analysis. For the pulse-chase, the cells were washed and incubated in starvation
medium (DMEM lacking methionine/cysteine (Thermo Scientific)) for 1 hour at 370C. They were
then labeled (pulse) by incubating them in starvation medium supplemented with 400 µCi of
[35S]methionine/cysteine (EasyTag™ EXPRESS35S Protein Labeling Mix, Perkin Elmer) per
plate for 20 min at 370C. After labeling, the cells were washed with complete DMEM (10% calf
serum) supplemented with 2mM L-Methionine and incubated (chase) at 370C in the same medium
for the indicated amount of time. Cells were collected at each time point and lysed in CelLytic M
cell lysis buffer (Sigma-Aldrich). MYC protein was immunoprecipitated from cell lysates
normalized by total labeled protein through Cerenkov counting using the c-Myc
Immunoprecipitation kit (Sigma-Aldrich) as per manufacturers instructions. Immunoprecipitates
were subjected to 10% polyacrylamide SDS-PAGE and autoradiography.
In vivo ubiquitination assays
HeLa cells were transiently transfected with the expression plasmids as indicated in the figure
legends for 18 hours. After 4 hours of treatment with 10 µM of MG132 proteosomal inhibitor, the
cells were either lysed with CelLytic M cell lysis buffer (Sigma-Aldrich) for MYC
immunoprecipitations or with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific)
for HA-Ubiquitin immunoprecipitations as per manufacturers instructions. The lysis and wash
buffers were supplemented with 5mM N-Ethylmaleimide (NEM) to inhibit deubiquitinase activity.
Cellular lysates were equalized for total protein concentration and used as whole cell extracts
(WCE) for immunoblots or for immunoprecipitating either MYC or HA-Ubiquitin using the Anti-
c-Myc Immunoprecipitation kit (Sigma-Aldrich) or Pierce@ Anti-HA Agarose (Thermo Scientific)
respectively, following the manufacturers instructions. Immunoprecipitates were subjected to
10% polyacrylamide SDS-PAGE and immunoblotting with the antibodies indicated in figure
legends.
In situ Proximity Ligation assays
For PLA, approximately 104 Hela cells were grown overnight in µ-Slide Angiogenesis (I-bidi).
PLA was conducted using the Duolink® In Situ PLA® Kit (Sigma) according to the
manufacturer’s protocol. The primary antibodies used were as follows: anti-BRD4 rabbit
polyclonal antibody was generated against the BRD4 C-terminal peptide (CFQSDLLSIFEENLF)
by a custom antibody service (Covance) (1:100 dilution), anti-MYC (9E10) mouse monoclonal
antibody (sc-40, Santa cruz biotechnology) (1:100 dilution), anti-ERK1 (G-8) mouse monoclonal
antibody (sc-271269, Santa cruz biotechnology) and anti-Nucleolin (sc-8031, Santa cruz
biotechnology) (1:100 dilution). Cells were observed with Zeiss LSM880 Multi-Photon Confocal
Microscope.
HAT assays
HAT assays were done as described previously (5) with minor changes. Purified BRD4 (500 ng),
or p300 (750 ng) was incubated with 1 μg of histone substrate (New England Biolabs), 0.6 mM
unlabeled Acetyl CoA (Sigma) in the presence of HAT buffer (10 mM butyric acid, 50 mM Tris
pH 8.0, 1 mM DTT, and 0.1 mM EDTA) at 30°C for 30 min. The reaction was stopped with SDS
sample buffer and the samples were run on 15% SDS-polyacrylamide gels that were
immunoblotted with the appropriate antibody as mentioned in the figure legends.
In vitro Nucleosome assembly
Purified 5S rDNA (208bp) was procured from New England Biolabs (NEB). Unmodified
recombinant human nucleosomes were assembled on the 5S rDNA with purified human histone
H2A/H2B dimers and histone H3/H4 tetramers using the EpiMark Nucleosome Assembly kit
(NEB) following the manufacturer’s instructions.
Nucleosome eviction assays
For in vitro eviction assays, 5S rDNA (7.7 pmoles) was end labelled with 20 pmoles of γ32P labeled
ATP using T4 polynucleotide kinase (NEB) according to the manufacturer’s protocol.
Nucleosomes were assembled in vitro on the radiolabeled 5S rDNA as described above. These
labeled nucleosomes were subjected to HAT assays under specific conditions as described in the
figure legend (Figure 5D). The release of free DNA upon nucleosome eviction was observed by
running the samples through a 6% polyacrylamide gel under native conditions and exposing it to
a phosphorimager screen.
In vitro Kinase assays
In vitro kinase assays with BRD4, ERK1 and with MYC or TAF7 were performed in 20 μl of
50mM Tris (pH 7.5), 5mM DTT, 5mM MnCl2, 5mM MgCl2 with 10μCi of γ32P ATP (6000Ci/mM)
and/or 40μM ATP where indicated in the figure legend. The kinase reactions for incubated for 1
hour at 300C, following which the proteins were resolved by SDS-PAGE and the extent of
phosphorylation quantitated by a phosphorimager. When MYC phosphorylation was determined
by immunoblotting with the MYC pThr58 antibody or subjected to Mass spectrometry as indicated
in the figures, kinase assays were performed with unlabeled ATP.
Mass spectrometric analysis of phosphorylated MYC
Purified recombinant MYC was subjected to a kinase assay with BRD4, ERK1 or no enzyme
(control) and run on an SDS PAGE gel. The phosphorylated MYC bands were excised from the
coommassie blue stained SDS-PAGE gel and subjected to in-gel tryptic digestion at 37C
overnight. Each digested peptide sample was desalted by C18 ZipTip (Millipore), lyophilized and
re-suspended in 16 µl of 0.1% formic acid and subjected to LC-MS analysis. Each sample (6 µl)
was then loaded on an Easy LC 1000 nano-capillary HPLC system (Thermo Scientific) with a 15
cm Acclaim® PepMap™ RSLC C18 column (Thermo Scientific) connected to an electrospray
ionization (ESI) emitter, coupled online with a dual-pressure linear ion trap (LTQ Velos Pro) mass
spectrometer (Thermo Scientific) for µRPLC-MS/MS analysis. The MS/MS data were searched
against the mouse MYC protein sequence using Proteome Discoverer 1.4 interfaced with
SEQUEST HT (Thermo Scientific). Up to two missed tryptic cleavage sites was allowed during
the database search. The results were normalized by deducting the phosphorylated peptides
detected in the no-enzyme control sample from those detected in the BRD4 and ERK1
phosphorylated samples.
Retrovirus generation
MSCV-Cre-eGFP retroviral construct was a gift from Dr. Jeff Zhu. The plasmid construct was
packaged into retrovirus in the Plat-E retroviral packaging cell line (Cell Biolabs inc) using the X-
tremeGENE 9 DNA transfection reagent (Sigma-aldrich) according to the manufacturer’s
protocol.
BRD4 KO mouse embryonic fibroblasts
Mouse embryonic fibroblasts with the Brd4 exon 3 flanked by 2 LoxP sites (floxed) were
developed as described previously (3). Cells were infected with the Cre-GFP bearing retrovirus
and sorted by FACS for GFP-positive cells to generate BRD4 deleted MEF cells. All experiments
with mice in the study were in compliance with the NIH animal care and use committee, approved
under protocol # EIB-076
Immunofluorescence analysis
BRD4 floxed MEFs uninfected or retrovirally infected with the Cre-GFP construct were FACs
sorted and plated on cover slips. The cells were fixed with 4% paraformaldeyhde, permeabilized
with 0.5% Triton-X, labeled with antibodies specific to MYC and MYC pThr58 and stained with
DAPI. Confocal image z-stacks were acquired using a Zeiss LSM510 META confocal microscope
using a 63x plan-apochromat (N.A. 1.4) objective lens, an optical slice thickness of 1.0 um, a x-y
pixel size of 0.349 um, and a z-step size of 0.5 um. The resultant image z-stacks were analyzed
using the Surpass module of Imaris (v. 7.7.1) software.
RNA-seq analysis
BRD4 floxed MEFs uninfected or retrovirally infected with the Cre-GFP construct were FACs
sorted and cultured for 5 days. Total RNA was isolated using TRIzol reagent (Invitrogen) following
the manufacturer's instructions. Reverse transcription was performed in a 20-μl reaction system
containing 1.5 μg of total RNA using oligo(dT) primer (Invitrogen) and the Thermoscript RT-PCR
system (Invitrogen) following the manufacturer's instructions to generate cDNA. RNA-seq
analysis was done as described before (5). The cDNA was fragmented to 100–200 bp using
sonication, followed by end repair and Solexa adaptor ligation. The products were sequenced on
an Illumina GAII system according to established procedures. RPKM values were calculated using
SeqGene package.
References
1. B. N. Devaiah et al., BRD4 is an atypical kinase that phosphorylates serine2 of the RNA
polymerase II carboxy-terminal domain. Proc Natl Acad Sci U S A 109, 6927-6932
(2012).
2. B. L. Allen-Petersen et al., Activation of PP2A and Inhibition of mTOR Synergistically
Reduce MYC Signaling and Decrease Tumor Growth in Pancreatic Ductal
Adenocarcinoma. Cancer Res 79, 209-219 (2019).
3. A. M. Baker et al., Distribution of the c-MYC gene product in colorectal neoplasia.
Histopathology 69, 222-229 (2016).
4. K. Suzuki, P. Bose, R. Y. Leong-Quong, D. J. Fujita, K. Riabowol, REAP: A two minute
cell fractionation method. BMC Res Notes 3, 294 (2010).
5. B. N. Devaiah et al., BRD4 is a histone acetyltransferase that evicts nucleosomes from
chromatin. Nat Struct Mol Biol 23, 540-548 (2016).
SUPPORTING FIGURES
Fig.S1. MYC binding site maps to 823-1044aa on BRD4.
A. BRD4 antibody specificity. WCE from Brd4-floxed MEFs uninfected (control) or
infected (BRD4 KO) with a Cre-GFP expressing retrovirus were immunoprecipitated
with anti-Myc antibody and immunoblotted with the indicated antibodies.
B. Top: Map of BRD4 segments used in pull down assays. Bottom: Graphs showing
quantification of MYC pulled down by BRD4 segments normalized to Flag beads
alone. Error bars represent ± SEM. (*, p<0.05).
Fig. S2. BRD4 phosphorylates MYC at Threonine 58 to maintain homeostatic levels of MYC
in vivo
A. BRD4 phosphorylates MYC. Autoradiograph of kinase assays with BRD4 (0.3 µg) or
equimolar amounts of BRD4 ∆kinase, ERK1 or GSK3β with or without 0.5 µg MYC
substrate.
B. MYC binds to both wild-type and kinase BRD4. Anti-MYC immunoblot of 0.2 µg
MYC recovered by pull-down with 0.5 µg wild type BRD4-Flag or equimolar amounts
of kinase BRD4 mutant on Flag beads. Anti-BRD4 immunoblot shows BRD4
retained on beads; beads alone and MYC input are shown as controls.
C. BRD4 phosphorylates MYC at its N-terminal end. Autoradiograph of kinase assays
with BRD4 (0.3 µg) using 0.5 µg of MYC or equimolar amounts of MYC 1-369aa, 1-
270aa and 1-63aa mutants as substrates.
D. BRD4 mediates exogenous MYC Thr58 phosphorylation in vivo. MYC pThr58, MYC,
BRD4 and tubulin immunoblots of whole cell extracts (WCE) from HCT116 (left
panel) and U2OS (right panel) cells transfected with empty vector (control) or 3µg
MYC with or without BRD4 (3µg).
E. The GSK3β inhibitors, CHIR-99021 and LY2090314, do not inhibit BRD4 kinase
activity at concentrations where GSK3β is inhibited. Equimolar amounts of
recombinant BRD4 (0.3 µg) and GSK3β were used in a kinase assay with 0.5 µg MYC
substrate with the indicated concentrations of GSK3β inhibitors or equal volumes of
DMSO.
F. BRD4 deletion does not alter MYC transcript levels in MEFs. Myc gene expression
profiles from RNA-seq analysis of BRD4 deleted and control MEFs. Analysis shows
gene expression profiles from two independent replicates (R1, R2). Blue boxes indicate
Myc exons.
G. BRD4 phosphorylation of endogenous MYC pThr58 does not depend on GSK3β in
MEFs. MYC pThr58, MYC, BRD4, GSK3β and tubulin immunoblots of WCE from
Brd4-floxed MEF cells uninfected (control) or infected (BRD4 KO) with a Cre-GFP
expressing retrovirus and treated with DMSO or GSK3β inhibitor CHIR-99021
overnight.
H. PTEFb does not phosphorylate MYC or affect BRD4 phosphorylation of MYC.
Autoradiograph of kinase assays done with BRD4 (0.3 µg), PTEFb (0.15 µg) or both
using 0.2 µg of MYC as substrate. The last lane shows a kinase assay where BRD4 (0.3
µg) pre-phosphorylated by PTEFb (0.15 µg) was used to phosphorylate 0.2 µg of MYC
in the presence of PTEFb inhibitor Flavopiridol (1 µM).
Fig. S3. Effect of BRD4 inhibitors on its regulation of MYC and BRD4 regulation of MYC
ubiquitination and stability.
A. JQ1 releases BRD4 from chromatin. BRD4 and Histone H3 immunoblots of chromatin
bound and chromatin free HeLa nuclear protein fractions from cells treated with either
500 nM JQ1 or DMSO.
B. JQ1 does not affect BRD4 binding to MYC in vitro. Anti- MYC and anti-BRD4
immunoblots of pull-down reactions done by incubating MYC (0.2 µg) with BRD4-Flag
(0.5 µg) on Flag beads in the presence of 1 µM JQ1 or DMSO.
C. JQ1 does not affect BRD4 phosphorylation of MYC. Autoradiograph of kinase assays
with BRD4 (0.3 µg) using 0.2 µg of MYC as substrate in the presence of 1 µM JQ1 or
DMSO.
D. Degradation of endogenous BRD4, but not its inhibition by JQ1 binding, leads to
endogenous MYC stabilization in MEF cells. MYC and BRD4 immunoblots of WCE from
MEF cells grown in the presence of either the PROTAC MZ1 (0.1 µM), 500 nM JQ1 or
DMSO (control) overnight, treated with 30 µg/ml cycloheximide (CHX) and harvested at
0, 30, 60, 90, and 120 mins after CHX treatment. Tubulin, loading control.
E. Degradation of endogenous BRD4 leads to MYC stabilization. Graphical representation
of autoradiographs of MYC immunoprecipitated from Brd4-floxed MEF cells uninfected
(control) or infected (BRD4 -/-) with a Cre-GFP expressing retrovirus and pulse chased
after labelling with [35S]methionine/cysteine for 20 min (pulse) and incubated in unlabeled
complete medium for the indicated times (chase).
F. BRD4 phosphorylates exogenous MYC Thr58 resulting in MYC ubiquitination. MYC
immunoprecipitated from whole cell extracts (WCE) of HeLa cells co-transfected with
4µg HA-ubiquitin and 3 µg of MYC, MYC T58A, MYC S62A and BRD4 as indicated
were immunoblotted with anti- ubiquitin-biotin, MYC or MYC pThr58 antibodies (upper
panels). Immunoblots of WCE with anti- BRD4 and Tubulin antibodies are shown as
controls (lower panel).
G. BRD4 phosphorylates exogenous MYC Thr58 resulting in MYC ubiquitination. HA-
Ubiquitin immunoprecipitated from whole cell extracts (WCE) of HeLa cells co-
transfected with 4µg HA-ubiquitin and 3 µg of MYC, MYC T58A and BRD4 as indicated
were immunoblotted with anti-MYC antibody, showing high molecular weight HA-
Ubiquitin bound MYC.
H. MAX co-immunoprecipitates with MYC and BRD4 but does not interact with BRD4
directly. Top: BRD4 was immunoprecipitated from HeLa nuclear extract using anti-BRD4
and immunoblotted with anti- MYC and MAX. Bottom: recombinant MAX (0.05, and
0.1µg) was pulled down with 0.5µg rBRD4 immobilized on Flag-beads and
immunoblotted with anti- MAX and BRD4 antibodies. MAX (0.1µg) input is shown as
control.
Fig. S4. MYC interacts with BRD4 and inhibits its HAT activity
A. MYC is not acetylated by BRD4. Anti-acetyl lysine and anti-MYC immunoblots of 0.2 µg
MYC subjected to HAT assays with 0.5 µg BRD4. (*, BRD4 autoacetylation).
B. MYC does not bind histone H3 directly or compete with BRD4 for H3 binding. Anti-
Histone H3, BRD4 and MYC immunoblots of pull-down reactions done by incubating
histone H3 (0.05 µg) with either MYC (0.2 µg) alone or with BRD4 (0.5 µg) on Myc-
agarose beads.
C. MYC does not reduce the efficiency with which BRD4 binds histone H3. Anti- MYC,
Histone H3 and BRD4 immunoblots of pull-down reactions done by incubating either
MYC (0.2 µg), histone H3 (0.05 µg) or both with BRD4-Flag (0.5 µg) on Flag beads.
Beads alone and the input proteins are shown as controls.
D. The BRD4 HAT domain is not required for MYC binding. Anti-MYC immunoblot of 0.2
µg MYC recovered by pull-down with 0.5 µg wild type His-BRD4-Flag or equimolar
amounts of His-BRD4-Flag ΔHAT on Flag beads. Anti-His immunoblot shows BRD4 on
beads and beads with or without MYC are shown as controls.
E. MYC inhibits acetylation of histone H3K122 in vivo. Histone H3K122ac, H3 and MYC
immunoblots of whole cell extracts (WCE) from U2OS cells transfected with 3µg murine
MYC or empty vector (control).
F. MYC inhibits histone H3K122 acetylation by BRD4 but not by p300. Histone H3K122ac
and H3 immunoblots of 1µg histone H3 subjected to HAT assays with BRD4 (0.5 µg) or
p300 (0.3 µg) with or without 0.2 µg MYC. H3K122ac levels (relative to the activity of
the HAT alone and normalized to total histone H3) are shown below the immunoblots.
G. MYC interacts with chromatin-bound BRD4. MYC immunoprecipitated from HeLa cells
treated with either 500nM JQ1 or DMSO for 4hrs was immunoblotted with anti- BRD4
and MYC antibodies. Relative levels of MYC bound BRD4, normalized to MYC, is shown.
H. BRD4 phosphorylation of MYC is attenuated by histone H3. Left: schematic of
experiment. Right: BRD4 (0.5 µg) pre-incubated alone or with 0.05 µg histone H3 for 20
minutes was used in a kinase assay with MYC (0.2 µg) and subjected to autoradiography.
In a third reaction, 0.05 ug histone H3 was added at the start of a kinase reaction with
BRD4 and MYC.
Fig. S5. ERK1 directly interacts with BRD4 and inhibits its kinase activity
A. ERK1 does not affect BRD4 HAT activity or MYC inhibition of BRD4 HAT activity.
Histone H3ac and H3 immunoblots of 1µg histones H3 subjected to a HAT assay with 0.5
µg BRD4 in the presence or absence of ERK1 (0.15 µg), MYC (0.2 µg) or both.
B. BRD4 and ERK1 are not phosphorylated by each other, but ERK1 inhibits BRD4
autophosphorylation. Autoradiograph of kinase assays done with BRD4 (0.5 µg) or
equimolar amounts of BRD4 Δkinase and ERK1 either alone or in combination.
C. ERK1 binding to BRD4 is compromised when BRD4 is histone bound. Anti- ERK1,
Histone H3 and His tag (BRD4) immunoblots of pull-down reactions done by incubating
either histone H3 (0.06 µg), ERK1 (0.15 µg) or both with His-BRD4-Flag ΔC (0.3 µg) on
Flag beads together or adding ERK1 after pre-incubating Histone H3 with BRD4 for 1 hr.
Beads alone and the input proteins are shown as controls.
Original immunoblot images of figures.
Fig. 2CkDa
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Anti-MYC pThr58 Anti-MYC
Anti-MYC pThr58 Anti-MYC
Anti-BRD4
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Fig. 2D
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Anti-MYC pThr58 Anti-MYC Anti-BRD4
Fig. 2E
Anti-MYC pThr58 Anti-MYC Anti-BRD4 Anti-GSK3
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Fig. 2F
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Anti-MYC pThr58 Anti-MYC Anti-MYC pSer62
Fig. 2G
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Anti-MYC pThr58 Anti-MYC Anti-BRD4
Fig. 2H
Anti-MYC pThr58 Anti-MYC Anti-BRD4
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Fig. 3ALeft panel
Right panel
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Anti-MYC pThr58 Anti-MYC pSer62 Anti-MYC Anti-BRD4 Anti-Tubulin
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Anti-MYC pThr58 Anti-MYC Anti-BRD4 Anti-Tubulin
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Anti-Ubiquitin Anti-MYC
Anti-MYC pThr58 Anti-MYC Anti-BRD4
Fig. 4ATop and middle panel Bottom panel