weili qu_sure2013 poster
Post on 14-Jan-2017
117 Views
Preview:
TRANSCRIPT
Results
Table 2. Summary of gene5c crosses So far, twenty crosses among various alleles of candidate Arabidopsis SWI/SNF genes were completed. Three double homozygous lines were obtained. There was no obvious phenotype observed from double homozygotes of taf14a x snf2a. However, there were unexpected phenotypes observed in both taf14a x snf5 and snf2e-‐1 x snf5 F2 plants where the phenotypes of both double homozygotes were not stronger than those of double heterozygotes or any other genotypic combinaEons. It is possible that these alleles are inducing inheritable epigeneEc alteraEons in F1 double heterozygotes. These alteraEons may then lead to unexpected phenotypic segregaEons in F2 populaEon that do not correlate with genotypes.
Functional Analysis of Chromatin Remodeling Enzymes in the Model Plant Arabidopsis thaliana
Weili Qu1, Paja Sijacic2 and Roger B. Deal2 1Emory College of Arts and Sciences, Emory University, Atlanta, GA 2Department of Biology, Emory University, Atlanta, GA
Abstract
� ATP-‐dependent chroma5n remodeling complexes (CRC) can alter the structure of
nucleosomes to allow access of condensed genomic DNA and regulate gene expression in eukaryoEc cells1. CRCs play an essenEal role during the development of mulEcellular organisms, which involves cell proliferaEon and differenEaEon. This requires precise control of transcripEon throughout the genome2. Since core subunits of these complexes are each encoded by small gene families, a given remodeler can have mulEple funcEonally disEnct isoforms in specific cell types1.
� SWI/SNF and SWR1 are mulEfuncEonal CRCs and are involved in key developmental
pathways in animals1. LiOle is known about the organizaEon and funcEon of putaEve plant SWI/SNF and SWR1 complexes although conserved SWI/SNF and SWR1 subunit homologues have been idenEfied3. In addiEon, it has been hypothesized that plants possess a large number of SWI/SNF-‐like isoforms given that the majority of the putaEve subunits are encoded by much larger and more diverse gene families than those of animals2.
� Nuclear Ac5n Related Proteins (ARPs) exist exclusively in CRCs3. ARP6 and ARP7 are core
subunits of SWR1 and SWI/SNF complexes, respecEvely, and are conserved in all eukaryotes. In Arabidopsis, ARP6-‐containing protein complexes funcEon as SWR1, while ARP7-‐containing protein complexes are likely to funcEon as CRCs and potenEally homologous to yeast SWI/SNF3.
Background
Figure 1. Phenotypic analysis of snf5 x snf2e-‐1 F2 plants There were unexpected phenotypes observed in snf2e-‐1 x snf5 F2 plants, where the phenotype of double homozygotes was not stronger than those of double heterozygotes or any other genotypic combinaEon. For instance, plants #2 and #5 are phenotypically different although they have the same genotype; plant #5, which is snf5 heterozygous and WT for snf2e-‐1, has stronger phenotype than plant #4, a snf5 homozygous and WT for snf2e-‐1.
Double heterozygous
Heterozygous for snf5, WT for snf2e-‐1
Homozygous for snf5, WT for snf2e-‐1
Heterozygous for snf5, WT for snf2e-‐1
12.78
7.18
11.45
7.19
0
2
4
6
8
10
12
14
16
WT arp6-‐1 not transformed
gARP6-‐FLAG in arp6-‐1
arp6-‐1 transformed
numbe
r of leaves a
t flow
ering
Figure 3. Screening for Arp6 rescued plants Plant #7 is a putaEve rescued plant, for it did not flower at the 6-‐leaf stage. Out of 1300 transformed plants, 45 restored WT flowering Eme and were considered rescued plants.
“rescued” plant “non-‐rescued” plant
Figure 4. Protein Blot Analysis of Arp6 rescued plants Plants 1 to 7 express FLAG-‐tagged ARP6 fusion protein, as indicated by the larger size of ARP6 protein bands compared to WT.
Conclusion and future direc5ons
� The unexpected phenotypes of snf5 x snf2e-‐1 and snf5 x taf14a F2 plants might be caused by epigeneEc interacEons among these alleles. AddiEonal geneEc interacEons among candidate genes of putaEve SWI/SNF complex will be analyzed.
� Arp6-‐FLAG transgene rescued arp6-‐1 plants and fusion protein was successfully detected on protein blot. Arp4 and Arp7 rescued plants will be generated. ImmunoprecipitaEon of tagged ARP4-‐, ARP6-‐, and ARP7-‐containing protein complexes will be opEmized, and the composiEon of ARP-‐containing protein complexes will be determined by MS/MS.
Figure 2. Flowering 5me analysis of Arp6 transgenic plants arp6-‐1 mutants have early flowering phenotype compared to WT (6-‐7 roseOe leaves for arp6-‐1 versus 12-‐14 roseOe leaves for WT at the Eme of flowering). Plants were considered rescued if flowering Eme was similar to WT, as determined by the average number of roseOe leaves at the Eme of flowering.
1 2 3 4 5 6 7 8 WT arp6-‐1
46 kDa—
WB: mAbARP6
The composi5on of SWI/SNF complex
Yeast SWI/SNF complex
AcEn related protein 7
DNA binding domain
Table 1. Puta5ve SWI/SNF homologues in Arabidopsis
Nucleosome, the repeaEng unit of eukaryoEc chromosome
ATP-‐dependent chromaEn remodeling complex
DNA binding protein
Gene that needs to be transcribed
hOp://www.ncbi.nlm.nih.gov/pmc/arEcles/PMC2924208/
hOp://www.nature.com/nature/journal/v463/n7280/full/nature08911.html
Acknowledgement: This material is based upon work supported by the Howard Hughes Medical InsEtute Science EducaEon Program award #52006923 to Emory University. Any opinions, findings, and conclusions or recommendaEons expressed in this material are those of the author(s) and do not necessarily reflect the views of the Howard Hughes Medical InsEtute or Emory University. A special thanks to the Deal Lab group for mentorship and research experEse.
References: 1. Hargreaves, Diana C., and Gerald R. Crabtree. "ATP-‐dependent ChromaEn Remodeling: GeneEcs, Genomics and Mechanisms." Cell Research 21.3 (2011): 396-‐420. 2. Ho, Lena, and Gerald R. Crabtree. "ChromaEn Remodeling during Development." Nature463.7280 (2010): 474-‐84. 3. Meagher, R. B. "Nuclear AcEn-‐Related Proteins as EpigeneEc Regulators of Development." Plant Physiology 139.4 (2005): 1576-‐585.
Yeast SWI/SNF genes Arabidopsis homologues
Methods
Single homozygous T-‐DNA seeds of candidate gene A Single homozygous
X
F1 Double heterozygous plant
Self-‐cross
F2 Double homozygous plant
PCR genotyping
Single homozygous T-‐DNA seeds of candidate gene B
PCR genotyping
Out-‐cross Examine and compare phenotypes, take pictures
PCR genotyping
Gel electrophoresis
DNA amplificaEon
Gene5c approach
Specific aim: to generate double homozygous lines and idenEfy geneEc interacEons among candidate genes of putaEve SWI/SNF complex. � Candidate genes were selected from a list of putaEve SWI/SNF homologs (Table 1.). � T-‐DNA mutant lines for candidate genes were obtained from Arabidopsis Biological
Resource Center (ABRC). � Double heterozygotes were generated by out-‐crossing single homozygous lines, and
double homozygotes were generated by self-‐crossing heterozygotes. Genotypes were confirmed by PCR.
� Phenotypes of double homozygotes were observed and compared with those of single homozygotes and WT to idenEfy geneEc interacEons among candidate genes.
Random T-‐DNA inserEons disrupt gene funcEon
Biochemical approach
Specific aim: to immunoprecipitate (IP) ARP-‐containing putaEve plant complexes, and determine the composiEon of IP-‐ed protein complexes by mass spectrometry (MS/MS). � Agrobacteria are used to introduce transgenes into mutant plants, and rescued lines
will be obtained through plate/phenotypic selecEon and PCR genotyping. � The expression of tagged ARPs in rescued plants will be confirmed by western blolng. � ARP-‐containing protein complexes will be purified from leaves by immunoprecipitaEon
and their composiEon will be determined by MS/MS analysis.
Transgene Fluorescent tag
gARP4 FLAG Introduced into
arp4 Ri
gARP6 FLAG Introduced into
arp6-‐1/arp6-‐1
gARP7 GFP Introduced into
arp7-‐1/ +
collect seeds
collect seeds
BASTA/anEbioEc selecEon on Petri dish
PCR genotyping
grow
Rescued plants
ARP-‐containing CRC Tag
AnEbody against Tag
MS Immu
noprecipita
Eon
Bead
AnEbody binds to protein, Non-‐binding proteins are removed by washing
Elute complex from beads
SDS-‐PAGE of eluted proteins Cut bands from SDS-‐PAGE gel, in gel
digesEon with trypsin then add idenEfy proteins by mass spectrometry (MS/MS)
hOp://www.proteome.org.au/NewsleOer-‐2012/Protein-‐Protein-‐interacEons-‐using-‐Mass-‐Spectrometry/default.aspx
hOp://www.intechopen.com/books/transgenic-‐plants-‐advances-‐and-‐limitaEons/transgenic-‐plants-‐as-‐gene-‐discovery-‐tools
hOp://www2.warwick.ac.uk/alumni/services/epormolios/hrrgak/project_overview/t-‐dna_screening_approach/
Single homozygous
In eukaryoEc cells epigeneEc pathways regulate the first step in gene expression— transcripEon, in which a parEcular segment of DNA is copied into RNA. ATP-‐dependent chromaEn remodeling complexes (CRCs) can alter the structure of nucleosome, the fundamental repeaEng unit of eukaryoEc chromaEn, and thus regulate gene expression. A class of ATP-‐dependent chromaEn remodeling complex called SWI/SNF plays an essenEal role in various developmental processes. Although well studied in fungi and animals, liOle is known about the funcEon and composiEon of SWI/SNF-‐like complexes in plants. This project aims to use the model plant Arabidopsis thaliana to study the funcEon and composiEon of plant SWI/SNF and SWI/SNF-‐related (SWR1) complexes using geneEc and biochemical approaches. Plants with mutant alleles for putaEve SWI/SNF subunits were crossed and phenotypes were examined to idenEfy geneEc interacEons among those alleles. In addiEon, biochemical techniques to purify and idenEfy subunits of SWI/SNF and SWR1 complexes are being established. This work will allow an understanding of the diversity and funcEon of SWI/SNF-‐like complexes in plant development.
top related