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Supplemental  Material  Supplemental  Materials  and  Methods  Supplemental  References  Table  S1.  Antibodies  and  primers  used  in  this  study  Figure  S1.  In  vivo  biotinylation  of  GATA4  Figure  S2.  PRC2  knockdown  by  shRNA  expressing  adenovirus  Figure  S3.  Inactivation  of  Ezh2fl  by  Nkx2-­‐5Cre  Figure  S4.  GATA4[K299R]  mutation  was  compatible  with  EZH2  and  DNA  binding  Figure  S5.  Cardiac  abnormalities  in  Ezh2NK  embryos.  Figure  S6.  The  small  molecule  DZNep  depleted  EZH2  and  Suz12  in  NRVMs.  Figure  S7.  Chromatin  occupancy  analysis  of  Myh6  regulatory  sequences.  Figure  S8.  PRC2  depletion  did  not  alter  GATA4  chromatin  occupancy  at  Myh6.  Figure  S9.  Effect  of  GATA4  methylation  on  GATA4  acetylation,  p300  binding,  and  chromatin  

recruitment.  Figure  S10.  Model  of  PRC2  regulation  of  cardiac  gene  expression.  

Materials  and  Methods  Mice.  Mice  with  Ezh2fl/fl  and  Nkx2-­‐5cre  alleles  were  described  previously  (Moses  et  al.,  2001;  

Shen  et  al.,  2008).  Gata4flbio  mice  were  generated  by  homologous  recombination  in  ES  cells,  resulting  

in  fusion  of  a  sequence  encoding  the  FLAG-­‐Bio  peptide  to  the  C-­‐terminus  of  Gata4.  A  Frt-­‐neo-­‐Frt  

cassette  was  placed  in  the  3’  UTR  and  subsequently  excised  by  breeding  through  Actb::Flpe  mice  

(Rodriguez  et  al.,  2000).  Gata4flbio/+  and  Rosa26BirA/BirA  mice  (Driegen  et  al.,  2005)  were  crossed  to  

obtain  Gata4flbio/flbio  Rosa26BirA/BirA  mice,  which  were  maintained  as  homozygotes.  Genotyping  was  

confirmed  by  PCR  of  tail  DNAs.  All  animal  experiments  were  performed  according  to  protocols  

approved  by  the  Institutional  Animal  Care  and  Use  Committee  of  Children’s  Hospital  Boston.  

Cell  Culture.  Neonatal  Rat  Ventricle  Myocytes  (NRVM)  were  isolated  from  rat  pups  at  day  1  as  

described  (Ikeda  et  al.,  2009).  HL1  cells  (Claycomb  et  al.,  1998)  were  a  gift  from  Dr.  Claycomb,  and  

cultured  in  Claycomb  Media  (Sigma).  Where  indicated,  NRVM  and  HL1  cells  were  treated  with  the  

PRC2  inhibitor  3-­‐deazaneplanocin  A  (DZNep  (Tan  et  al.,  2007))  for  72  hr.  HL1  cells  were  

transfected  in  6  well  dishes  using  Lipofectamine  2000  (Invitrogen)  following  the  manufacturer’s  

protocol.  Briefly,  2  µg  of  DNA  and  6  µl  of  Lipofectamine  2000  were  used,  with  total  DNA  kept  

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constant  by  adding  empty  pcDNA3.1+  vector  as  needed.  The  Dual  Luciferase  Assay  (Promega)  was  

performed  according  to  the  manufacturer’s  protocols.  The  GATA4  expression  construct  and  

multimerized  GATA  luciferase  construct  were  described  previously    (Durocher  et  al.,  1997;  

Chandrasekar  et  al.,  2005).  

Chromatin  Immunoprecipitation.  E12.5  heart  ventricle  or  HL1  cells  were  used  for  chromatin  

immunoprecipitation  as  described  (He  and  Pu,  2010).  ChIP  grade  antibodies  used  were:  EZH2  

(#3147,  Cell  Signaling),  EZH2  (17-­‐662,  Millipore),  H3K27me3  (17-­‐622,  Millipore),  GATA4  (sc-­‐

1237x,  Santa  Cruz),  and  p300  (sc-­‐585x,  Santa  Cruz).  In  brief,  DNA  levels  were  first  normalized  to  

the  internal  control  region  in  the  first  intron  of  Actb.  Relative  enrichment  was  calculated  by  dividing  

the  normalized  level  of  ChIPed  DNA  to  that  of  input  DNA  at  the  corresponding  locus.  ChIP-­‐qPCR  

results  were  reported  as  mean  ±  SD  for  at  least  three  ChIP  biological  replicates.  Primer  sequences  

are  provided  in  Supplemental  Table  1.  

Protein-­‐protein  interactions.  Cells  were  rinsed  in  PBS,  then  nuclei  were  isolated  by  

resuspending  in  Hypotonic  Lysis  Buffer  (20  mM  HEPES  pH  7.5,  10  mM  KCl,  1  mM  EDTA,  0.1  mM  

Na3VO4,  0.1  mM  0.2%  (vol/vol)  Nonidet  P40  (NP-­‐40),  10%  (vol/vol)  glycerol  plus  protease  

inhibitor  cocktail  (Roche).  Embryonic  hearts  were  homogenized  in  PBS  and  then  processed  as  cells.  

Nuclei  were  then  resuspended  in  lysis  buffer  (50  mM  Tris-­‐HCl  pH  8.0,  150  mM  NaCl,  0.5%  Nonidet  

P-­‐40,  1  mM  EDTA,  and  fresh  1  mM  PMSF  and  protease  inhibitor  cocktail.  Insoluble  material  was  

removed  by  centrifugation  at  16,000  x  g  for  20  min  at  4°C,  yielding  nuclear  extracts.    Where  

indicated,  lysates  were  treated  with  20  U  of  Benzonase  nuclease  (Stratagene)  for  1  hr  at  4°C  before  

centrifugation.  Nuclear  extracts  were  precleared  with  Protein  A  beads,  then  incubated  with  primary  

antibody  for  16  hr  at  4°C  followed  by  Protein  A  beads.  For  biotinylated  protein  precipitation,  

extracts  were  incubated  with  SA  beads  (M-­‐280  beads,  Invitrogen)  for  4  hr  at  4°C.  Beads  were  

washed  four  times  (10  min  each)  with  the  lysis  buffer.  Precipitated  proteins  were  recovered  by  

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boiling  in  sample  buffer.  GATA4  and  EZH2  immunoprecipitation  used  antibodies  listed  under  

“Chormatin  Immunoprecipitation”  above.  FLAG  immunoprecipitation  used  immobilized  FLAG  

antibody  (Sigma).  

Western  Blot.  Cell  lysates  were  prepared  in  lysis  buffer.  Equal  amounts  of  total  protein  (50-­‐80  

µg)  were  resolved  on  4%-­‐15%  SDS-­‐polyacrylamide  gels  and  immunoblotted  with  primary  

antibodies  to  EZH2  (#4905,  Cell  Signaling,  1:1000),  SUZ12  (39357,  Active  Motif,  1:2000),  EED  (a  

kind  gift  from  D.  Reinberg,  1:1500)  (Margueron  et  al.,  2009),  mono/di-­‐methyl-­‐Lysine  (ab23366,  

Abcam,  1:500),  GAPDH  (RDI-­‐TRK5G4-­‐6C5,  Fitzgerald  Industries,  1:10,000).  Immunoreactive  bands  

were  visualized  by  HRP-­‐conjugated  secondary  antibodies  (Goat  anti-­‐mouse  and  Goat  anti-­‐rabbit  

IgG,  Invitrogen)  and  detected  by  enhanced  chemiluninescence  (Millipore).    

Mass  spectrometry  identification  of  methylated  residues  of  GATA4.  E16.5  

Gata4flbio/flbio,Rosa26BirA/BirA  and  GATA4+/+  Rosa26BirA/BirA  heart  ventricles  were  extracted  with  SDS  

lysis  buffer  (50  mM  Tris-­‐HCl  pH  8.0,  150  mM  NaCl,  2%  SDS,  1  mM  EDTA,  and  fresh  1  mM  PMSF  and  

protease  inhibitor  cocktail),  precleared  with  protein  A  beads,  and  incubated  with  SA  beads  for  2  hr  

at  4°C.  After  washing  twice  in  2%  SDS  and  twice  in  high  salt  buffer  (50  mM  Tris-­‐HCl  pH  8.0,  500  mM  

NaCl,  0.5%  Nonidet  P-­‐40,  1  mM  EDTA,  and  fresh  1  mM  PMSF  and  proteases  inhibitors  cocktail),  

bound  protein  complexes  were  subjected  to  SDS-­‐PAGE.    

The  Gata4flbio  band  was  excised,  digested  with  sequencing  grade  trypsin  (Promega),  and  

analyzed  in  an  LC/MS  system  consisting  of  a  micro-­‐autosampler,  Suvery  HPLC  pump  and  an  LTQ  

mass  spectrometer  (all:  Thermo  Scientific,  San  Jose,  CA).  Mass  spectrometric  data  was  searched  

against  the  international  protein  index  (IPI  mouse  v3.39)  database  using  the  protein  identification  

software  Mascot  (v2.2.04,  Matrix  Sciences,  London,  UK).    Search  criteria  included  tryptic  peptide  

specificity  with  one  missed  cleavage,  Carbamidomethyl  (C)  as  a  fixed  modification,  Deamidated  

(NQ),Gln-­‐>pyro-­‐Glu  (N-­‐term  Q),  Oxidation  (M),  Methyl  (KR),  Dimethyl  (KR),  and  Trimethyl  (KR)  as  

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variable  modifications  for  MS2  spectra.    A  1.5  Da  peptide  tolerance  for  the  MS  and  a  0.8  Da  peptide  

tolerance  for  MS/MS  spectra  were  implemented  along  with  a  mascot  ion  cut  off  score  of  30.  

 GST  fusion  proteins.  pDest15  (Invitogen)  derived  expression  plasmids  were  generated  

encoding  fragments  of  mouse  GATA4  fused  to  GST  were  transformed  into  BL21(DE3)  (Stratagene).  

Protein  expression  and  purification  were  performed  as  described  (Harper  and  Speicher,  2008)  

In  vitro  translation  and  pull-­‐down  assay.  Plasmids  for  in  vitro  expression  of  EZH2,  EED,  

SUZ12,  GATA4-­‐FLAG  and  NKX2-­‐5-­‐FLAG  were  created  in  pcDNA-­‐Dest40  (Invitrogen)  using  the  

Gateway  cloning  system.  The  linearized  plasmids  were  in  vitro  transcribed  and  translated  using  the  

TNT  T7  Coupled  Reticulocyte  Lysate  Kit  (Promega)  in  the  presence  of  35S-­‐methionine.  In  vitro  

translated  proteins  were  mixed  with  GATA4-­‐FLAG  in  binding  buffer  (25  mM  Tris-­‐HCl  (pH  7.5),  150  

mM  NaCl,  1  mM  dithiothreitol,  0.05%  Nonidet  P-­‐40)  and  co-­‐immunoprecipitated  with  immobilized  

FLAG  M2  antibody  (Sigma).  After  4  washes  in  the  same  buffer,  bound  proteins  were  loaded  onto  a  

4%-­‐15%  SDS-­‐polyacrylamide  gel  and  detected  by  autoradiography.  

   Peptides  G4P1bio  and  G4P1mebio  were  chemically  synthesized  by  Abgent  according  to  murine  

GATA4  residues  249-­‐323  and  contains  an  N-­‐terminal  biotin  modification.  The  methylated  form  

contains  a  monomethyl-­‐lysine  corresponding  to  K299. For the protein-protein interaction assays,

1 µg of peptide was incubated with in vitro translated proteins and pulled-down using

streptavidin beads.  

PRC2  Methyltransferase  assay.  Recombinant  PRC2  was  purified  from  over-­‐expressing  

baculovirus  as  described  previously  (Shen  et  al.,  2008).  Methyltransferase  assays  were  performed  

on  5  µg  of  GATA4  proteins  or  peptides  as  described  (Shen  et  al.,  2008).  

Gene  Expression  Analysis.  RNA  was  purified  from  the  ventricular  apex  of  embryonic  hearts  

using  the  RNeasy  kit  (Qiagen)  with  on  column  DNase  treatment.  After  reverse  transcription  with  

SuperScript  III  (Invitrogen),  quantitative  PCR  reactions  were  performed  using  the  POWER  SYBR  

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master  mix  (ABI)  and  an  ABI  7500  real  time  thermal  cycler.  Primer  sequences  are  provided  in  

Supplemental  Table  1.  qPCR  data  were  reported  as  mean  ±  SEM.  

Immunohistochemistry.  Tissues  were  fixed  in  4%  paraformaldehyde  for  1  hr  at  4°C,  and  

processed  for  immunohistochemistry  as  described  (Zhou  et  al.,  2009).  Images  were  acquired  on  an  

Olympus  FV1000  confocal  microscope.    

Goat  Troponin  I  (TNNI3)  (ab56357,  Abcam)  was  used  at  1:500.  The  GATA4-­‐K299me  antibody  

was  produced  by  Yenzyme  against  the  peptide  GLYM-­‐Kme1-­‐LHGVPRPLC.  Methylated  GATA4-­‐

specific  antibody  was  purified  by  immunodepletion  of  antibody  that  bound  the  unmethylated  

peptide.  For  immunostaining,  the  antibody  was  used  at  1:200  dilution.  

DNA-­‐binding  assay.  DNA  binding  reactions  were  performed  as  described  (Dodou  et  al.,  2004).  

The  oligonucleotides  used  for  EMSA  are  provided  in  Supplementary  Table  1.  

Plasmids  and  Adenovirus.  The  pGL3-­‐Myh6  promoter  plasmid  was  created  by  placing  the  5.5  

kb  Myh6  promoter  (Gulick  et  al.,  1991)  upstream  of  firefly  luciferase  in  SmaI-­‐digested  pGL3-­‐basic  

(Promega).  Plasmids  expressing  EZH2,  EED  and  SUZ12  were  generated  by  inserting  the  

corresponding  cDNA  into  pcDNA3.1+  containing  the  Gateway  attR1/attR2  cassette  downstream  of  

the  CMV  promoter.  The  inducible  adenovirus  system  for  expression  of  GATA4flbio  was  described  

previously  (He  and  Pu,  2010).  Adenovirus  expressing  either  shRNA  against  Suz12  or  scrambled  

control  sequence  was  generated  as  described  (Bisping  et  al.,  2006).  The  Suz12  and  Eed  shRNAs  

targeted  the  sequences  GCTGTTACCAAGCTCCGAG  and  GCTATCAATGAGCTGAAATTC  respectively,  and  

the  scrambled  control  sequence  was  AGGTTAGACCGACAGGAGAA.

GATA4K299me  specific  antibody.  The  GATA4-­‐K299me  antibody  was  generated  in  rabbits  

inoculated  with  a  K299  monomethylated  GATA4  peptide  (Yenzyme)  and  depleted  against  non-­‐

methylated  GATA4  peptide.  

  6  

Statistics.  Results  are  displayed  as  mean  ±  SEM,  except  for  ChIP  data,  which  are  displayed  as  

mean  ±  SD.  Statistical  testing  was  performed  using  the  student’s  t-­‐test.  P  <  0.05  was  considered  

statistically  significant.  

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Supplemental  Material  References  Bisping,  E.,  S.  Ikeda,  S.W.  Kong,  O.  Tarnavski,  N.  Bodyak,  J.R.  McMullen,  S.  Rajagopal,  J.K.  Son,  Q.  Ma,  

Z.  Springer,  P.M.  Kang,  S.  Izumo  and  W.T.  Pu.  2006.  Gata4  is  required  for  maintenance  of  

postnatal  cardiac  function  and  protection  from  pressure  overload-­‐induced  heart  failure.  Proc  

Natl  Acad  Sci  U  S  A  103:  14471-­‐14476.  

Chandrasekar,  B.,  S.  Mummidi,  W.C.  Claycomb,  R.  Mestril  and  M.  Nemer.  2005.  Interleukin-­‐18  is  a  

pro-­‐hypertrophic  cytokine  that  acts  through  a  phosphatidylinositol  3-­‐kinase-­‐phosphoinositide-­‐

dependent  kinase-­‐1-­‐Akt-­‐GATA4  signaling  pathway  in  cardiomyocytes.  J  Biol  Chem  280:  4553-­‐

4567.  

Claycomb,  W.C.,  N.A.J.  Lanson,  B.S.  Stallworth,  D.B.  Egeland,  J.B.  Delcarpio,  A.  Bahinski  and  N.J.J.  Izzo.  

1998.  HL-­‐1  cells:  a  cardiac  muscle  cell  line  that  contracts  and  retains  phenotypic  characteristics  

of  the  adult  cardiomyocyte.  Proc  Natl  Acad  Sci  U  S  A  95:  2979-­‐2984.  

Dodou,  E.,  M.P.  Verzi,  J.P.  Anderson,  S.M.  Xu  and  B.L.  Black.  2004.  Mef2c  is  a  direct  transcriptional  

target  of  ISL1  and  GATA  factors  in  the  anterior  heart  field  during  mouse  embryonic  

development.  Development  131:  3931-­‐3942.  

Driegen,  S.,  R.  Ferreira,  A.  van  Zon,  J.  Strouboulis,  M.  Jaegle,  F.  Grosveld,  S.  Philipsen  and  D.  Meijer.  

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factors  Nkx2-­‐5  and  GATA-­‐4  are  mutual  cofactors.  Embo  J  16:  5687-­‐5696.  

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Protein  Sci  Chapter  6:  Unit  6.6.  

He,  A.  and  W.T.  Pu.  2010.  Genome-­‐wide  location  analysis  by  pull  down  of  in  vivo  biotinylated  

transcription  factors.  Curr  Protoc  Mol  Biol  Chapter  21:  Unit  21.20.  

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Ikeda,  S.,  A.  He,  S.W.  Kong,  J.  Lu,  R.  Bejar,  N.  Bodyak,  K.H.  Lee,  Q.  Ma,  P.M.  Kang,  T.R.  Golub  and  W.T.  

Pu.  2009.  MicroRNA-­‐1  negatively  regulates  expression  of  the  hypertrophy-­‐associated  

calmodulin  and  Mef2a  genes.  Mol  Cell  Biol  29:  2193-­‐2204.  

Margueron,  R.,  N.  Justin,  K.  Ohno,  M.L.  Sharpe,  J.  Son,  W.J.r.  Drury,  P.  Voigt,  S.R.  Martin,  W.R.  Taylor,  

V.  De  Marco,  V.  Pirrotta,  D.  Reinberg  and  S.J.  Gamblin.  2009.  Role  of  the  polycomb  protein  EED  in  

the  propagation  of  repressive  histone  marks.  Nature  461:  762-­‐767.  

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Supplementary Table 1. Primers used in this study.

ChIP-qPCR primersprimer ID Forward Reverse NoteIntergenic Control ATTTTGTGCTGCATAACCTCCT TAGCAACATCCTAAGCTGGACA Negative controlActb CGTATTAGGTCCATCTTGAGAGTACACAGTATTGCCATTGAGGCGTGATCGTAGC ReferenceMyh6-1 GAAGTGAGAAATGGGTGGAAAG CGTCTTGGTTTATCTTGGCTCTMyh6-2 ATGGGCAGATAGAGGAGAGACA CAGTTGTTCAACTCACCCTTCAMyh6-3 AGGAACACTCTCCCTGCTACC CCTTGGGGAGACACCATATTACMyh6-4 ATATGTCACTGCCTGGTTCTCA AAGCTGACCCAATGTTCTCAGTMyh6-5 ATCTTGAGGCTCTACCACCAGT AAGGAGATGTGTGGAGAAGTCCNeurog1 TGGTCTCCTGAGTGATGTCG GCCGTACTTAAGGGGTCCTG Ezh2, H3K27me3 pos controlSmarcd3 CCGCATTCCTGCACTGATA ACGCGGAGTGCACAGGAG Gata4 pos controlAnkrd1 GTGGTCACTGCCAAAGGAAT CCAAGAGGGAGATGACAAGCAtp1a1 GCTGTGCTGACTCCATAGCA GAGAGCAGGGCACTTGAATCCol4a3 CCAGTCAAGCATTCCACAGA AGACAACAGGGGCATTTCAGCsrp3 TAAGAGGCAGGCCAAAGCTA AGCCCCACCAAGTATGTGTCCtnna1 TGACCACAGCAGAAAACGAG GACGACTGTGGTTCACAGGADisc1 TGACCTGAGACTTGGGCTCT TGTGGGTCCTGTGTTTTTGADkk GCCTGGCATTCGAAATAAGT GGGCATTCCTCACTAAAGCAFgf12 TCACCCTAGCTGCCTTCTGT AGTGCCACACTTCTGCTCCTFos GAATGGAGAATGGGAAGCAG TCCACGCCTTTATCTTCTGGIgf2r CTACTCAGCTCTGCCCCTGT CTGCCACGTACATAGGCTCAKcnj5 AGGGGCTCTTCTCCATCAGT AACCCATGTGTGCCATTTCTMyl7 CGTGCCTGCCAGAGAAGTAT ACGGATTCAGTGGGAGTCAGMyom2 GCCTTGCCCTTCTCTCTAGC TGTCACCTCAGAATGCAAGGPGC GGGGAACCTGTGGAATTTTT AAGAGCCTGGTTAGGGGAGAPparg TTCCCCTTTCTCCTCTGTGA TTTTAAGGACGGTTGGTGGAPrkaa2 GTAAAGTCTGGCATGCACCA TGTACGGAGCATTGGAGAGAPrkag2 CCAGGCCACAGAGTCAGAGT CCCCTGTGACCAGCAAGTATRor1 TGATCAAGCTTGTCTGTCAGG CCTCCTATCTGTGTCTGCCTCTRyr2 AGAGGCCATTCTGGGTAGGT CCTGTGTGCTGACAGGGTTATbx18 CACCAAGGCATTTTCAACAG CCCAGTGTTCCATCTCTTCATbx5 CAAACCATATCCCCACCAGA TGGCCACTATCGAAATGACA

RT-qPCR primersGene Forward Reverse SpeciesMyh6 ACGGTGACCATAAAGGAGGA TGTCCTCGA TCTTGTCGAAC mouseMyh6 (Rat) TCAAACTGGAGCTGGATGAC GTATTCATTGGCCTGGTCCT ratEzh2 TTACTGCTGGCACCGTCTGATGTG TGTCTGCTTCATCCTGAGAAATAATCTCCmouseGapdh 4308313, Applied BiosystemsDkk3 GAGATGTTTCGAGAGGTGGAG TTGTGATAGTTGGGAGGTAAGC mousePrkag2 AGGCACCGGAATTAACTTCTAG CTCTTCAGGCTAGGGTTCTTTT mouseFgf12 TTGCAGCTTCCAGACTCAG CGGTCCCTTTAGAGTGCTG mouseMyl7 ATCAACTTCACCGTCTTCCTC ACTCTTCCTTGTTCACCACC mouse

Primers for cloning mouse Myh6 promoterMyh6-promoter-F cggggtacc ATCCTGCAAGGTCACACAAGGGTCTMyh6-promoter-R GACTCAAACTCTTATGGGGGAGATAG

Primers for gateway cloning Gata4 truncation into pDest15.G4F1-F-attB1 gggg aca agt ttg tac aaa aaa gca ggc t tg atg tac caaa gcctggccatgG4F1-R-attB2 gggg ac cac ttt gta caa gaa agc tgg gt tta tc tgccttctga gaagtcatcG4F2-R-attB2 gggg ac cac ttt gta caa gaa agc tgg gt tta ggc aca ggagagg cctacccG4F3-R-attB2 gggg ac cac ttt gta caa gaa agc tgg gt tta cttccg tttt ctggtttgG4F4-R-attB2 gggg ac cac ttt gta caa gaa agc tgg gt tta cgc ggtgattatg tccccat

EMSA GATA4 probewt top strand GCAACGTGCAGCCGGAGATAAGACCCGGCTCTAGAGGmut top strand GCAACGTGCAGCCGGAGTGCAGACCCGGCTCTAGAGG

A

B

D

rtTA BirAIRES TRE

rtTAdox

Gata4 Bio

GATA4

biotin

3’ probe5’ probe

wt

chim

era

1 2 3 4 5

PupsE

fb/fb

fb/+

+/+

n=93

F

Frequencyat Weaning 0.0

1.0

2.0

3.0

fb/+ fb/fb

LVEDD

fb-neowt

fb-neowt

G

0.0

0.2

0.4

0.6

FS

fb/+ fb/fb

H

GAPDH

GATA4G4flbio

G4wt

Dox

BirA

Gat

a4flb

io

inSA beadsC

Gat

a4flb

io

IB: Flag

Supplemental Fig. S1, He et al. In vivo biotinylation of GATA4. A. Expression system to express and biotinylate GATA4 in HL1 cells. We established an adenovirus that expresses the reverse tet activator protein (rtTA) and BirA, an E. coli enzyme that recognizes and biotinylates the 15 amino acid Bio sequence. A second adenovirus expressed GATA4 fused to FLAG and Bio epitope tags. B. Dox titration to establish GATA4flbio expression at near endogenous levels. C. GATA4flbio was biotinyl-ated by BirA in HL1 cells. Protein lysates prepared from HL1 expressing BirA alone or BirA plus GATA4flbio were precipitated on immobilized streptavidin. Precipitates were probed with FLAG anti-body. GATA4flbio was retained on the streptavidin beads, indicating biotinylation. D. Proper homolo-gous recombination in ES cells was confirmed by Southern Blotting. Arrowhead indicates the wild-type allele, and arrow indicates the targeted allele. E. Germline transmission (arrows) of the targeted Gata4flbio-neo allele was confirmed by PCR. F. After removal of the Frt-neo-Frt cassette by ActB::Flpe, Gata4flbio/+ mice were intercrossed. Gata4flbio/flbio mice survived normally. G-H. Gata4flbio/flbio mice had normal ventricular size and function by echocardiography at one month of age. n=2. Red dots indicate individual data points.

SUZ12

EZH2

H3K27me3

Gapdh

H3K4me3C

ontro

l

shS

uz12

shS

uz12

shS

uz12

Con

trol

Con

trol

25 50 100 MOIshRNAVirus

Imm

unob

lot

Supplemental Fig. S2, He et al.PRC2 knockdown by shRNA-expressing adenovirus. A. Adenovirus delivering shRNA against murine Suz12 depleted SUZ12 from HL1 cells. EZH2 was also depleted, consistent with loss of Suz12-stabilizing activity. H3K27me3 was reduced with high degree of SUZ12 and EZH2 knockdown. Control adenovirus contained a scrambled sequence shRNA. B. Adenovirus delivering shRNA against murine Eed depleted EED from HL1 cells. Bracket indicates viral dose used for subsequent experiments. C. Eed shRNA significantly reduced GATA4 lysine methylation. The left panel shows a repre-sentative immunoblot. GATA4flbio-expressing HL1 cell nuclear extract was precipitated on streptavidin beads. GATA4flbio lysine methylation was assessed with meK antibody. Precipitated GATA4 was assessed with FLAG antibody. The right panel shows quantita-tion of three independent experiments.

A

me-K

FLAG

Ctrl sh-Eed

G4flbio SA IP

0

1.0

Rel

ativ

e m

eKIm

mun

orea

ctiv

ity

Ctrl

sh-E

ed

P<0.05

sh-E

ed

Con

trol

Eed

Gapdh

Adenovirus

sh-E

ed

Con

trol

sh-E

ed

Con

trol

B

IBC

IB

Con

trol

A B

EZH

2TN

NI3

Ezh

2NK

00.20.40.60.81.01.2

Ezh2fl/+

Nkx2-5

Cre/+

Ezh2fl/fl

Nkx2-5

Cre/+

**

Ctrl Ezh2NK0

0.5

1.0

1.5G

ata4

mR

NA

D TNNI3 GATA4 DAPI

Ctrl Ezh2NK

181310

181310

G4P

1

G4P

1-K

299m

e

G4P

1

G4P

1-K

299m

e

GATA4 (total)Antibody

GATA4-K299meAntibody

kD kD

C E

Supplemental Fig. S3.He et al. Inactivation of Ezh2fl by Nkx2-5Cre. A. Relative Ezh2 expression by qRTPCR in E9.5 heart with heterozygote or homozygote Ezh2 inactivation by Nkx2-5Cre. B. Immunohistochemistry of control and Ezh2NK E12.5 heart. Cardiomyocyte EZH2 expression (arrowheads) was strongly decreased in mutants. Non-myocyte expression (arrows) appeared intact. Bar = 50 µm. C. Validation of antibody specific for GATA4-K299me. GATA4-K299me antibody was highly specific for methylated GATA4. No signal was detectable for unmethylated GATA4. 1 ng of each peptide was used for immunoblotting. D. Gata4 mRNA was unchanged in E16.5 control or Ezh2NK mutant heart. E. Total GATA4 expression in myocytes (arrowheads, top) and non-myocytes (arrows, top) was not altered by EZH2 mutation.

35S EZH2

G4C

G4C

[K29

9R]

207

441

207

441

AG4C

K299RG4C

probe

No

prot

einB

Supplemental Fig. S4, He et al.GATA4[K299R] mutation was compatible with EZH2 and DNA binding. a. GST-G4C[K299R] and GST-G4C were incubated with in vitro synthesized, S35-labeled EZH2 and pulled down on glutathione beads. Co-precipitated proteins were analyzed by SDS-PAGE, and EZH2 was detected by autoradiography. b. GST-G4C[K299R] retained DNA binding activity comparable to GST-G4C in EMSA assay. The protein-DNA complex migrated as two bands (arrowhead and arrow). The ratio of the two bands differed between G4C and G4C-K299R, sug-gesting a difference in conformation of the protein-DNA complex.

A

B

RVRV

RARA

LALA

LVLV

Ezh2NKCtrl

E16.5 E16.5

Ezh2NK

LVRV

RARA

E16.5 E16.5

Ctrl

Supplemental Fig. S5. He et al.Cardiac abnormalities in Ezh2NK embryos. H&E stained transverse sections of E16.5 embryos revealed thinning of compact myocardium, more pronounced in RV than LV (black arrowheads), excessive myocardial trabeculation (blue arrowheads), right atrial dilation, membranous and muscular VSDs (double headed arrows), and atrial septal defect (dashed double headed arrow). Blue arrow indicates the normal foramen ovale. Bar=500 μm.

IB

DZNep (µM)0 50 5

* EZH2

SUZ12

GAPDH

Supplemental Fig. S6. He et al.The small molecule DZNep depleted EZH2 and Suz12 in NRVMs. Arrow indicates full lengh EZH2. Asterisk indicates a proteolytic product of EZH2.

0

5

10

Ezh

2 E

nric

h (R

b A

b)

0

10

20A

ctb

inte

rgen

icP

os C

trl

1 2 4 53

H3K

27m

e3E

nric

hG

ATA

4flbio

Enr

ich

Myh6

0

10

20

1 kbsite:

0

20

40E

zh2

Enr

ich

(Ms

Ab)

Supplemental Fig. S7. He et al.Chromatin occupancy analysis of Myh6 regulatory sequences. ChIP-qPCR analysis of E12.5 heart ventricle chromatin. GATA4 occupied site 5 of the Myh6 promoter/enhancer. EZH2 and H3K27me3 were not substan-tially enriched. Positive control was Neurog1 for EZH2 and H3K27me3 and Smarcd3 for GATA4. Bars in GATA4 indicate independent biological repli-cates. Bars for EZH2 and H3K27me3 indicate mean ± SD (n=3).

G4 249-323 K299 K299-Me K299

Competitor – – – – – – wt

mut–

No

probe

A

GA

TA4flb

io

Enr

ichm

ent

Ctrl shSuz12

10

1 2 3 4 5Myh6 site

5

0

B

Supplemental Fig. S8. He et al.PRC2 depeletion did not alter GATA4 chromatin occupancy at Myh6 regula-tory elements. A. Electrophoretic mobility shift assay showed that GATA4[249-323] peptide specifically bound DNA, yielding a complex (arrow) that was elimi-nated by 100-fold excess wild-type unlabeled competitor, but not unlabeled mutated competitor. K299 methylation reduced in vitro DNA-binding affinity of the peptide. B. ChIP-qPCR measurement GATA4 occupancy of Myh6 regulatory sites. SUZ12 knockdown did not significantly alter GATA4 occupancy of Myh6 regulatory elements in HL1, as determined by ChIP-qPCR. The numbered sites correspond to the sites mapping in Supplemental Fig. 7. n=3. C. ChIP-qPCR measurement of GATA4 occupancy of regulatory sites of other genes. Primers were designed against binding sites identified by GATA4 ChIP-seq (He et al, 2011). SUZ12 knockdown did not significantly alter GATA4 chromatin occupancy at these sites. n=3. NS, not significant.

C

0102030405060

Inte

rgen

ic c

trl

Ank

rd1

Ryr

2

Dkk

3

Myl

7

Prk

ag2

Fgf1

2

GAT

A4flb

io

enric

hmen

t

Ctrl shSuz12

PR

C2

knoc

kdow

nNS NS NSNS NS NS

NS NS NS NS NS

GST-G4C

GST

IB: F

lag

Col

Blu

e

p300

EZH2

p300PRC2

GST GSTG4C+ ++

– ++++

– +

GSTK299RA

0102030405060708090

100110120130

Act

b

Ank

rd1

Atp

1a1

Col

4a3

Csr

p3

Ctn

na1

Dis

c1 Fos

Igf2

r

Kcn

j5

Myo

m2

PG

C1-

v

Ppa

rg

Prk

aa2

Ror

1

Ryr

2

Tbx1

8

Tbx5

B

p300

ChI

PFo

ld E

nric

hmen

t

GAT

A4

knoc

kdow

n

Ctrl Gata4 shRNA* **†*** * *

Supplemental Fig. 9. He et al.Effect of GATA4 methylation on GATA4 acetylation, p300 binding, and chromatin recruitment. A. In vitro GATA4-p300 interaction assay. Indicated GST-GATA4 fusion proteins were immobilized on glutathione beads and incubated with recombinant p300. Preincubation with PRC2 and SAM reduced p300 binding. Blocking GATA4 methylation with the K299R mutation abrogated the effect of PRC2. This mutant binds PRC2 but cannot be methylated by it. B. p300 Chip-qPCR from HL1 cells treated with control or GATA4 shRNA adenovirus. GATA4 knockdown significantly reduced p300 recruitment to chromatin at 12 of 21 tested loci (*, P<0.05; see also Fig. 4J). At the remaining loci, there was no effect at 8 and increased p300 recruitment at one (†, P<0.05). Test loci were sites with co-occurrence of GATA4 and p300 by ChIP-seq (He et al., 2011). n=3. C. GATA4 K299R mutation blocked GATA4 acetylation by p300 in vitro. Although K299 is not a major site of GATA4 acetylation, mutation to arginine blocked GATA4 acetylation in vitro by p300.

C

ColBlue

14C-Ac

GST-G4C wt K299R wt K299R

PRC2p300 +

– – + ++ ++

EED Suz12

H3K27me3

TAD CTD

ZnF-CZnF-NBR

K299

GATA4

PRC2

EZH2

TAD CTD

K299me

p300

TAD CTD

AcAc

targetgene

Supplemental Fig. S10. He et al.Model of PRC2-regulation of cardiac gene expression. PRC2 represses gene transcription by establishing H3K27me3 epigenetic marks, or by direct methylation of transcription factors. Activity of the cardiac transcription factor GATA4 was regu-lated by the balance between PRC2-mediated methylation of K299 and the p300-mediated acetylation of the GATA4 C-terminal domain (CTD). TAD, transcriptional activation domain. Nu, nucleosome. ZnF, zinc finger. BR, basic region. Ac, acetyla-tion. Me, methylation.

Nu Nu Nu

targetgene