PRC2 directly methylates GATA4 and represses its transcriptional activity

Abstract

Polycomb-repressive complex 2 (PRC2) promotes tissue-specific differentiation by depositing trimethylated histone H3 Lys 27 (H3K27me3) epigenetic marks to silence ectopic gene expression programs. Here, we show that EZH2, the catalytic subunit of PRC2, is required for cardiac morphogenesis. Both in vitro and in fetal hearts, EZH2 interacted with cardiac transcription factor GATA4 and directly methylated it at Lys 299. PRC2 methylation of GATA4 attenuated its transcriptional activity by reducing its interaction with and acetylation by p300. Our results reveal a new mechanism of PRC2-mediated transcriptional repression in which PRC2 methylates a transcription factor to inhibit its transcriptional activity.

Figure 1.

EZH2 interacted with GATA4. (A) GATA4^flbio, expressed in HL1 cells and precipitated on SA beads in the presence of benzonase, coprecipitated PRC2 components EZH2, EZH1, SUZ12, and JARID2. ASH2, WDR5, and RbBP5 were not detected in the precipitate. (B) EZH2, immunoprecipitated from HL1 cells expressing GATA4^flbio, coprecipitated GATA4. Biotinylated GATA4 was detected with SA-HRP. (C) EED, EZH2, SUZ12, and Flag-tagged GATA4^flbio, synthesized and ³⁵S-labeled in vitro, were Flag-immunoprecipitated. EZH2 specifically interacted with GATA4. (D) Chemically synthesized G4P1^bio peptide (murine GATA4 residues 249–323) coprecipitated ³⁵S-labeled, in vitro translated EZH2 on SA beads. (E) Gene targeting strategy for generation of GATA4^flbio knock-in mice. C-terminal Flag and bio epitope tags were placed on the GATA4 C terminus. (F) Nuclear extracts from GATA4^flbio/flbio Rosa26^BirA/BirA or GATA4^+/+ Rosa26^BirA/BirA (control) E16.5 embryo hearts were incubated with SA beads. Coprecipitated EZH2 was detected by immunoblotting. (G) Nuclear extract from wild-type E16.5 embryo hearts was incubated with GATA4 antibody or IgG (control). Coprecipitated EZH2 was detected by immunoblotting.

Figure 2.

GATA4-K299 was methylated in vivo. (A) GATA4 was methylated in HL1 cells. GATA4^flbio, precipitated from GATA4^flbio-expressing or control HL1 extracts in the presence of 2% SDS, immunoreacted with meK-specific antibody at the appropriate molecular weight for GATA4^flbio. No meK immunoreactivity was detected in parallel control experiments with Tbx5^flbio. (B) GATA4 methylation in E16.5 hearts. Protein lysates from Gata4^flbio/flbio Rosa26^BirA/BirA or Gata4^+/+ Rosa26^BirA/BirA (control) hearts were analyzed as in A. (C) Identification of GATA4 methylation site. GATA4^flbio/flbio Rosa26^BirA/BirA or control E16.5 heart protein lysates were precipitated as in A. (Inset) SDS-PAGE of precipitated proteins confirmed GATA4 pull-down (colloidal blue-stained). Tandem mass spectrometry identified GATA4 methylation at K299. The mass spectrum of one diagnostic peptide is shown. Fragments containing the original N-terminal amino acid (marked by asterisks) did not show mass shift from a methyl residue, while fragments containing the original C-terminal amino acid (marked by open circles) were shifted by 14 Da. Lines in the peptide sequence indicate observed N-terminal and C-terminal fragment ions.

Figure 3.

PRC2 directly methylated GATA4. (A) Suz12 shRNA knockdown inhibited methylation of GATA4^flbio in HL1 cells. GATA4^flbio methylation was assessed as in Figure 2, A and B. The arrowhead indicates the position of the GATA4^flbio band. (B) E16.5 heart cryosections stained for cardiac marker TNNI3 and K299-methylated GATA4. GATA4-K299me immunoreactivity (arrowheads) was markedly reduced in Ezh2^NK mutant cardiomyocytes. (C) Recombinant PRC2 methylated GATA4 fragments that encompass K299. GATA4 was chemically synthesized (G4P1^bio, residues 249–323) or purified as GST fusion proteins from bacteria. GATA4 residues in the constructs are G4P1^bio, 249–323; G4C, 207–441; G4FL, 1–441; and G4N, 1–214. After incubation with PRC2 and ³H-SAM, reaction products were resolved by SDS-PAGE and detected by autoradiography. Histones were used as positive control. (D) Mutation of GATA4-K299 abrogated methylation by PRC2.

Figure 4.

PRC2 modulated GATA4 activation of Myh6. (A) Myh6 transcripts were up-regulated in EZH2-deficient E12.5 and E16.5 fetal hearts. (B) PRC2 knockdown by DZNep up-regulated endogenous Myh6 transcript in NRVMs. (C) Suz12 knockdown in HL1 up-regulated the endogenous Myh6 transcript. (D) GATA4 activation of Myh6-luc was blocked by transfection of PRC2 components Ezh2, Eed, and Suz12 and by K299R mutation. (E) Suz12 knockdown in HL1 up-regulated activity of Myh6-luc and potentiated the effect of GATA4 overexpression. (F,G) PRC2 abolished p300 stimulation of GATA4 transcriptional activity on Myh6-luc and 3xGATA-luc reporters. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Figure 5.

PRC2 antagonized p300 acetylation of and binding to GATA4 in vitro and in vivo. (A) In vitro GATA4 acetylation by p300 was markedly attenuated by preincubation with PRC2 and the methyl donor SAM. Acetylation of GST fusion proteins was detected by p300-mediated labeling by ¹⁴C-acetyl-CoA. Coom indicates Coomassie blue-stained gel, which showed equal protein loading. (B) Quantitation of A. Numbers correspond to groups indicated in A. (C) PRC2 regulates GATA4 acetylation in HL1 cells. GATA4^flbio-expressing HL1 cells were treated with control or Suz12 shRNA. GATA4^flbio was then pulled down and probed with acetyl lysine or meK antibodies. (Open arrowhead) Methylated GATA4; (closed arrowhead) acetylated GATA4. (D) PRC2 regulates p300 recruitment by GATA4 in HL1 cells. p300 occupancy at indicated loci was determined by ChIP-qPCR in the context of GATA4 or PRC2 knockdown. (E) Effect of PRC2 on mRNA expression in HL1, as measured by qRT–PCR. (*) P < 0.05; (**) P < 0.01; (NS) not significant.