Jarid2 (Jumonji, AT rich interactive domain 2) regulates NOTCH1 expression via histone modification in the developing heart

Abstract

Jarid2/Jumonji, the founding member of the Jmj factor family, critically regulates various developmental processes, including cardiovascular development. The Jmj family was identified as histone demethylases, indicating epigenetic regulation by Jmj proteins. Deletion of Jarid2 in mice resulted in cardiac malformation and increased endocardial Notch1 expression during development. Although Jarid2 has been shown to occupy the Notch1 locus in the developing heart, the precise molecular role of Jarid2 remains unknown. Here we show that deletion of Jarid2 results in reduced methylation of lysine 9 on histone H3 (H3K9) at the Notch1 genomic locus in embryonic hearts. Interestingly, SETDB1, a histone H3K9 methyltransferase, was identified as a putative cofactor of Jarid2 by yeast two-hybrid screening, and the physical interaction between Jarid2 and SETDB1 was confirmed by coimmunoprecipitation experiments. Concurrently, accumulation of SETDB1 at the site of Jarid2 occupancy was significantly reduced in Jarid2 knock out (KO) hearts. Employing genome-wide approaches, putative Jarid2 target genes regulated by SETDB1 via H3K9 methylation were identified in the developing heart by ChIP-chip. These targets are involved in biological processes that, when dysregulated, could manifest in the phenotypic defects observed in Jarid2 KO mice. Our data demonstrate that Jarid2 functions as a transcriptional repressor of target genes, including Notch1, through a novel process involving the modification of H3K9 methylation via specific interaction with SETDB1 during heart development. Therefore, our study provides new mechanistic insights into epigenetic regulation by Jarid2, which will enhance our understanding of the molecular basis of other organ development and biological processes.

FIGURE 1.

H3K9 methylation is altered in Jarid2 KO mice. Jarid2 KO hearts have significantly decreased levels of H3K9me2 and H3K9me3 on the specific region of the Notch1 genomic locus. Quantitative ChIP was performed on E17.5 WT (gray bar) or Jarid2 KO (black bar) mouse hearts with antibodies specific for H3K9me1 (A), H3K9me2 (B), or H3K9me3 (C). No differences were observed in Jarid2 KO hearts for H3K27me1 (D), H3K27me2 (E), or H3K27me3 (F). *, p < 0.05 using a Student's paired t test. Error bars represent mean ± S.E.M.

FIGURE 2.

Jarid2 physically interacts with SETDB1. A, diagram depicting the protein structures of Jarid2 and SETDB1. Jarid2 contains a JmjN, AT-rich interacting domain (ARID), JmjC, and zinc finger (ZF) domain. SETDB1 contains a Tudor, Methyl CpG (MBD) binding domain and a bifurcated Set domain. A yeast two-hybrid using Jarid2 as bait identified cDNA encoding aa 979–1307 of SETDB1 as the region mediating physical interaction. B, coimmunoprecipitation was performed on E17.5 heart extracts. Lanes 1–3 indicate input, immunoprecipitate (IP) with non-specific IgG or Jarid2 antibody, respectively, followed by immunoblotting (IB) with SETDB1 antibody. Lanes 4–6 indicate input, IP with IgG or SETDB1 antibody, respectively, followed by IB with Jarid2 antibody. C–H, immunostaining was performed on E17.5 sections. Brown deposits indicate expression of Jarid2 (C and F) or SETDB1 (D and G). No brown deposits were detected when non-specific IgG was used (E and H). Arrows indicate endocardial cells, and arrowheads indicate myocardial cells. Scale bars = 100 μm (C and F). RV, right ventricle; VS, ventricular septum; LV, left ventricle.

FIGURE 3.

GST pull-down assays determine domains mediating interaction between Jarid2 and SETDB1. A, various [³⁵S]methionine-labeled Jarid2 mutant proteins (lanes 1–4) were incubated with GST alone (lanes 5–8) or with GST-SETDB1 (lanes 9–12) containing only the bifurcated SET domain. Jarid2 Ct and Jarid2 DBD bound to GST-SETDB1 SET only. B, [³⁵S]methionine-labeled Jarid2 Ct was incubated with GST (lane 1), GST-SETDB1 FL (lane 2), GST-SET only (lane 3), and GST-no SET (lane 4). GST-SETDB1 FL and GST-SET only bound to Jarid2 Ct. +, binding; -, no binding.

FIGURE 4.

Jarid2 is required for SETDB1 accumulation at the +1150 bp region of the Notch1 locus. A, Jarid2 KO mice have significantly decreased accumulation of SETDB1 at the Notch1 locus. Quantitative ChIP was performed on E17.5 WT (gray bar) or Jarid2 KO (black bar) mouse hearts using a SETDB1-specific antibody. B, proposed model of Jarid2 regulation of Notch1 expression. The expression of Jarid2 is required for the recruitment of SETDB1 to the +1150 bp region of the Notch1 locus, resulting in methylation (indicated by the asterisk) of H3K9 and Notch1 silencing. Deletion of Jarid2 results in failed recruitment of SETDB1 and no methylation of H3K9.

FIGURE 5.

Genome-wide analysis on the promoter occupancy by Jarid2, SETDB1, and H3K9me3. A, Venn diagram demonstrating the overlap of genome-wide occupancy of Jarid2, SETDB1, and H3K9me3 by ChIP-chip. B, graph showing the number of genes dysregulated more than 1.2-fold when the ChIP-chip was overlapped with the microarray. C, representative SignalMap peak display for a gene occupied (Notch1) or unoccupied (Lztfl1, Leucine zipper transcription factor-like 1) by Jarid2, SETDB1, and H3K9me3. D, table of up-regulated ChIP-chip genes that overlap with up-regulated biological processes identified by microarray analyses. PNPLA6, patatin-like phospholipase domain containing 6; GRN, granulin, SNED1, Sushi, nidogen, and EGF-like domains 1; CLDN7, Claudin 7; ROR2, receptor tyrosine kinase-like orphan receptor 2, ITGA11, integrin α 11; CLSTN1, Calsyntenin 1; NPPB, natriuretic peptide type B.