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.