Jarid2 (Jumonji, AT Rich Interactive Domain 2) RegulatesNOTCH1 Expression via Histone Modification in theDeveloping Heart*
Received for publication, October 21, 2011, and in revised form, November 17, 2011 Published, JBC Papers in Press, November 21, 2011, DOI 10.1074/jbc.M111.315945
Matthew R. Mysliwiec‡, Clayton D. Carlson§, Josh Tietjen§, Holly Hung‡, Aseem Z. Ansari§, and Youngsook Lee‡
From the ‡Department of Cellular and Regenerative Biology and the §Department of Biochemistry and The Genome Center ofWisconsin, University of Wisconsin-Madison, Madison, Wisconsin 53706
Background: Jarid2 regulates Notch1 expression in the developing heart through an unidentified mechanism.Results: Regulation of Notch1 by Jarid2 is through recruitment of SETDB1, resulting in increased methylation of histone H3lysine 9.Conclusion: Jarid2 regulation of a subset of genes during cardiac development involves histone methylation through SETDB1recruitment.Significance: This is a novel mechanism of epigenetic regulation by Jarid2 during cardiac development.
Jarid2/Jumonji, the foundingmember of the Jmj factor family,critically regulates various developmental processes, includingcardiovascular development. The Jmj family was identified ashistone demethylases, indicating epigenetic regulation by Jmjproteins. Deletion of Jarid2 in mice resulted in cardiac malfor-mation and increased endocardial Notch1 expression duringdevelopment. Although Jarid2 has been shown to occupy theNotch1 locus in the developing heart, the precise molecular roleof Jarid2 remains unknown. Here we show that deletion ofJarid2 results in reduced methylation of lysine 9 on histone H3(H3K9) at theNotch1 genomic locus in embryonic hearts. Inter-estingly, SETDB1, a histone H3K9methyltransferase, was iden-tified as a putative cofactor of Jarid2 by yeast two-hybrid screen-ing, and the physical interaction between Jarid2 and SETDB1was confirmed by coimmunoprecipitation experiments. Con-currently, accumulation of SETDB1 at the site of Jarid2 occu-pancywas significantly reduced in Jarid2knock out (KO)hearts.Employing genome-wide approaches, putative Jarid2 targetgenes regulated by SETDB1 via H3K9 methylation were identi-fied in the developing heart by ChIP-chip. These targets areinvolved in biological processes that, when dysregulated, couldmanifest in the phenotypic defects observed in Jarid2 KOmice.Our data demonstrate that Jarid2 functions as a transcriptionalrepressor of target genes, includingNotch1, through a novel proc-ess involving the modification of H3K9 methylation via specificinteraction with SETDB1 during heart development. Therefore,our study provides newmechanistic insights into epigenetic regu-lation by Jarid2, which will enhance our understanding of themolecular basis of other organ development and biologicalprocesses.
Jarid2 is required for normal cardiac development, and allmice harboring a homozygous Jarid2 deletion (Jarid2 KO) diein the uterus or right after birth (1–3). We have previouslyreported that whole body or endothelial-specific deletion ofJarid2 (Jarid2en) results in cardiac defects mimicking humancongenital cardiac defects, including ventricular septal defects,double outlet right ventricle, and hypertrabeculation associatedwith noncompaction of the ventricular wall resulting in a thincompact layer (1, 4, 5). Notch1 signaling is critical for normalcardiac development. Whole body or endothelial deletion ofNotch1 in the mouse results in embryonic lethality at embry-onic day 10.5 (E10.5)2 with hearts showing little or no trabecu-lation (6, 7).We identifiedNotch1 as a potential target of Jarid2and observed elevated Notch1 expression in the endocardiumand elevated Notch1 signaling to the underlying myocardiumin Jarid2 KO and Jarid2en embryonic hearts (5). This dysregu-lation of the Notch1 pathway is a potential cause for the defectsobserved. However, the precise mechanistic function of Jarid2in regulation of Notch1 expression in the developing heartremains to be elucidated.Histone methylation was once considered to be static and an
enzymatically irreversible chromatin modification. However,recent reports have shown that bothmethylation and demethy-lation of histones is a highly regulated process that allows forfine epigenetic regulation of many cellular processes includingtranscriptional regulation, regulation of cell fate, and cell pro-liferation (8). For example, methylation of H3K9 and H3K27 isgenerally associatedwith gene silencing (9–11). Jarid2 has beenreported to function as a transcriptional repressor and to inter-act with other nuclear factors (4, 5, 12–17). Jarid2 is the found-ing member of the Jumonji family of proteins, all of whichcontain the JmjC domain that generally confers histonedemethylase activities. The recent discovery of Jmj family fac-tors as histone demethylases has ushered in a new era of inves-tigating the role of histone methylation status in regulating
* This work was supported, in whole or in part, by National Institutes of HealthGrants HL067050 (to Y. L.) and NIGMS069420 (to A. Z. A.). This work wasalso supported by American Heart Association Grant 10POST2600279 (toM. R. M.) and predoctoral fellowship 0615615Z (to C. D. C.), and by U.S.National Human Genome Research Institute (NHGRI) training grant to theGenomic Sciences Training Program (GSTP) 5T32HG002760 (to J. T.).
1 To whom correspondence should be addressed: 1300 University Ave., SMI321, Madison, WI 53706. Fax: 608-262-7306; E-mail: [email protected].
2 The abbreviations used are: E10.5, embryonic day 10.5; H3K27, histone H3lysine 27; PRC, polycomb repressor complex; H3K9me2, dimethyl H3K9;H3K9me3, trimethyl H3K9; aa, amino acids; DBD, DNA binding domain.
gene expression. Intriguingly, Jarid2 contains substitutions atkey amino acids necessary for enzymatic function and is highlylikely enzymatically inactive (18–20). Therefore, transcrip-tional regulation by Jarid2 may be dependent on binding part-ners that function as histone modifiers. Recent work suggeststhat Jarid2 is involved in methylation of histone H3 lysine 27(H3K27) through interaction with members of the polycombrepressor complex (PRC) in embryonic stem cells, induced plu-ripotent stem cells, and in epidermal stem cells (20–25).Although Jarid2 is uniformly agreed to interact with the PRCcomplex and to be crucial for normal differentiation of embry-onic stem cells, the precise role of Jarid2 in regulation of histonemethylation status is conflicting. Most importantly, it remainsto be determined whether Jarid2 interacts with any histone-modifying enzymes to regulate cardiac morphogenesis in thedeveloping heart. Therefore, it is imperative to delineatewhether dysregulation of gene expression in Jarid2 KOmice isdue to improper epigenetic regulation via defective histonemodification.To identify the molecular mechanisms by which Jarid2 reg-
ulates target gene expression in the developing heart, we inves-tigated the regulation of Notch1 by Jarid2, focusing on themethylation status of lysine residues of histone H3 at theNotch1 locus. We provide evidence that Jarid2 directly regu-lates Notch1 expression through interaction with a specificenhancer region of the Notch1 locus. Our study indicates thatdeletion of Jarid2 results in decreased dimethyl and trimethylH3K9 (H3K9me2 and H3K9me3) at the same region occupiedby Jarid2 on the Notch1 locus, which correlates well with aber-rant continued Notch1 expression in the Jarid2 KO hearts. Weshow that Jarid2 interacts with the H3K9 methylase SETdomain, bifurcated 1 protein (SETDB1) (26). Further, Jarid2 isrequired for the recruitment of SETDB1, which confersH3K9me2 and H3K9me3 at the Notch1 enhancer region. Thisdefect in histone modification likely causes failure to regulateNotch1 expression, contributing to the defects observed inJarid2 mutant hearts. Finally, we have performed ChIP-chipexperiments on the developing heart for Jarid2, SETDB1, andH3K9me3, and identified a critical subset of genes regulated byJarid2 and SETDB1whose dysregulationmay be involved in thephenotypic defects observed in Jarid2 KO hearts. Therefore,our current study provides new insights into epigenetic regula-tion of cardiac development by Jarid2, whichwill form a basis toinvestigate other organ development and broad biologicalprocesses.
EXPERIMENTAL PROCEDURES
Quantitative ChIP—Quantitative ChIP was performed asdescribed (5, 27) at least three times using two pooled E17.5hearts for each experiment. Antibodies used were H3K27me1(Upstate, catalog no. 07-448), H3K27me2 (Abcam, catalog no.ab24684), H3K27me3 (Abcam, catalog no. ab6002), H3K9me1(Upstate, catalog no. 05-1248), H3K9me2 (Upstate, catalog no.07-441), H3K9me3 (Upstate, catalog no. 07-442), SETDB1(Abcam, catalog no. ab12317, and Santa Cruz Biotechnology,Inc., catalog no. sc66884). Recovered DNA was used for quan-titative real-time PCR in triplicate using the standard curvemethod with primers described previously (5).
Yeast Two-hybrid Screening—Full-length Jarid2 was used asbait in a yeast two-hybrid screen to identify cofactors asdescribed (15). Recovered plasmids from growing colonieswere subjected to confirmation mating experiments, and thecDNAs were identified by sequencing and BLASTing (BasicLocal Alignment Search Tool) against the National Center forBiotechnology Information GenBankTM. Among 25 indepen-dent positive clones, SETDB1 was identified.Protein Detection and Protein-Protein Interaction—Coim-
munoprecipitation was performed as described (4, 12, 15) withminor modifications. Briefly, precleared nuclear extracts fromE17.5 hearts in 11 mM Tris-HCl (pH 8.0), 1.1 mM EDTA (pH8.0), 11% glycerol, 0.2% SDS, and 1 mM DTT, and proteinaseinhibitors were diluted 1:5 with 20mMTris-HCl (pH 8.0), 2mM
EDTA (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.01% SDS, 1mM DTT, and proteinase inhibitors and immunoprecipitatedwith nonspecific rabbit IgG or antibodies against Jarid2 (14, 15)or SETDB1 (Santa Cruz Biotechnology, Inc., catalog no.sc66884), followed by incubation with protein A agarose beads,SDS-PAGE, and Western blotting.Immunostaining was performed on 15-�mE17.5 sections as
described (5) with minor modifications. Following antigenretrieval in 10 mM sodium citrate (pH 6.0) and peroxidasequenching, sections were blocked in avidin and biotin block(Thermo Scientific) and BS buffer (PBS plus 0.2% TritonX-100, 1% glycine, 1% BSA, 5% normal goat serum). Sectionswere incubated in BS buffer with antibodies against Jarid2(14, 15), SETDB1 (Santa Cruz Biotechnology, Inc., catalogno. sc66884), or normal rabbit IgG, followed by incubationwith a HRP-linked secondary antibody. AEC chromogen sin-gle solution (RTU, Thermo Scientific) was used for develop-ment, followed by counterstaining with hematoxylin. Imageswere taken using a Zeiss Axiovert 200 microscope and anAxioCam HRc camera.To map the protein-protein interaction domains, GST pull-
down assays were performed as described (12, 15). All Jarid2constructs used were described previously (12, 14). Full-lengthmouse SETDB1 (aa 1–1307), SETDB1 containing the SETdomain only (aa 813–1307), and SETDB1 lacking the SETdomain (aa 1–812) were cloned into pcDNA3.1-myc-HisB(-)(Invitrogen) and pGEX-2T (Amersham Biosciences). [35S]me-thionine-labeled proteins, including Jarid2 C terminus (Ct) (aa529–1234), Jarid2 N terminus (Jarid2 Nt, aa 1–528), the Jarid2DNAbinding domain (Jarid2DBD, aa 529–792), and the Jarid2JmjC domain (aa 807–1234) were incubated in NETN buffer(100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.5%Nonidet P-40) with GST-glutathione beads or GST-SETDB1SET-only beads. For the reciprocal experiment, 35S Jarid2 Ctwas incubated with GST beads, GST-SETDB1 full-length(SETDB1 FL, aa 1–1307), GST-SET domain only (GST-SETonly, aa 813–1307), or GST-no SET domain (GST-no SET, aa1–812).Animal Husbandry and Genotyping—All mice were housed
in accordance with University of Wisconsin Research AnimalResource Center policies. Jarid2male and female heterozygousmice in amixed 129/Svj and C57BL/6 genetic backgroundweremated to produce Jarid2 KOmice. Genotyping was performedas described (1).
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ChIP-chip Assays—ChIP-chip was performed as described(28). Briefly, sonicated chromatin from 20 pooled E17.5 fixedhearts was immunoprecipitated using Jarid2, SETDB1, orH3K9me3 antibodies, followed by reversal of cross-linking andDNApurification. Immuno-enrichedDNA targets were ampli-fied by whole genome amplification appropriate to final yieldand fluorescently labeled using ligation-mediated PCR. Thelabeled fragments were hybridized onto the Roche NimbleGen3X720K RefSeq promoter arrays and scanned with an Axon4000B. These arrays cover the promoters of the well character-ized RefSeq genes. After the arrays were extracted using Nim-blescan (Roche), global and local normalization and datasmoothing in R was performed, and peaks were detected usingChIPOlte (29) and in-house algorithms. Peaks with a p valueless than 10�14 were used for analysis.
RESULTS
Jarid2 Deletion Results in Decreased Methylation of H3K9—Wehave shownpreviously that Jarid2 occupies a specific regionof the Notch1 genomic locus in E17.5 wild type (WT) embry-onic hearts, likely resulting in Notch1 repression (5). However,the molecular mechanism of Jarid2 in regulation of Notch1expression has not been elucidated. Histonemethylation statusis a crucial determinant of gene regulation. H3K9me2 andH3K9me3 are commonly associated with heterochromatin for-
mation and gene silencing. Interestingly, we have identifiedSETDB1,which functions as aH3K9methylase as a Jarid2 bind-ing protein by yeast two-hybrid screening. We therefore inves-tigated whether Jarid2 occupancy at theNotch1 locus is associ-ated with regulation of H3K9 methylation, specificallycomparing WT and Jarid2 KO mouse hearts at E17.5 whenNotch1 is normally repressed. The levels of methylation wereexamined on the Notch1 locus at both Jarid2-occupied andJarid2-unoccupied regions by ChIP assays using antibodiesagainst H3K9me1, H3K9me2, and H3K9me3 (5). There was nosignificant difference in the levels of methylation of H3K9me1between the WT and Jarid2 KO mouse (Fig. 1A). In contrast,both H3K9me2 (Fig. 1B) and H3K9me3 (C) were enriched atthe �1150 bp site in the WT but significantly reduced in theJarid2 KO heart. This suggests that Notch1 is repressed in theWT mouse because of accumulation of methylation of H3K9and that loss of Jarid2 disrupts this epigenetic mechanism,resulting in a failure ofNotch1 down-regulation in the develop-ing heart.Jarid2 has recently been shown to recruit the PRC in embry-
onic stem cells, which functions tomethylate H3K27 that act asa repressivemark. Therefore, we investigatedwhether the dele-tion of Jarid2 also resulted in a change of methylation at H3K27on theNotch1 locus. ChIP onWTor Jarid2KOE17.5 hearts for
FIGURE 1. H3K9 methylation is altered in Jarid2 KO mice. Jarid2 KO hearts have significantly decreased levels of H3K9me2 and H3K9me3 on the specificregion 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 forH3K9me1 (A), H3K9me2 (B), or H3K9me3 (C). No differences were observed in Jarid2 KO hearts for H3K27me1 (D), H3K27me2 (E), or H3K27me3 (F). *, p � 0.05using a Student’s paired t test. Error bars represent mean � S.E.M.
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H3K27me1 (Fig. 1D), H3K27me2 (E), orH3K27me3 (F) showedno significant difference at the Notch1 locus. This result sug-gests that H3K27 methylation is not involved in the regulationof Notch1 expression by Jarid2 in late-stage developing hearts.Jarid2 Physically Associates with SETDB1 in Vivo—In an
effort to determine physical interactor(s) of Jarid2, we per-formed yeast two hybrid screening (15) and identified SETDB1as a potential cofactor of Jarid2 (Fig. 2A). The recovered cDNAfrom yeast encoded the second bifurcation of the SET domain(aa 979–1307), suggesting that this region of SETDB1 is suffi-cient to interact with Jarid2 in yeast.It is important to examine whether both Jarid2 and SETDB1
are expressed in the developing heart, as the expression patternof SETDB1 is unknown in themouse embryonic heart.Wehaveconfirmed that both factors are expressed in the embryonichearts by Western blotting (Fig. 2B, lanes 1 and 4). To deter-mine whether Jarid2 and SETDB1 interact in vivo, coimmuno-precipitation experiments were performed on heart extracts.Whenheart extractwas immunoprecipitatedwith a Jarid2 anti-body, SETDB1 protein was detected (Fig. 2B, lane 3). SETDB1
was not detected when a nonspecific mouse IgG was used forimmunoprecipitation (Fig. 2B, lane 2), indicating the specificinteraction of Jarid2 with SETDB1. The converse experimentemploying a SETDB1 antibody for immunoprecipitation alsorevealed the specific interactions of Jarid2 with SETDB1 (Fig.2B, lane 6). Jarid2 was not detected when a nonspecific IgGwasused for immunoprecipitation (Fig. 2B, lane 5). These dataclearly demonstrate that Jarid2 physically associates withSETDB1 in vivo in the developing hearts. To confirm that Jarid2and SETDB1 are both expressed in the same cell lineageswithinthe developing heart, immunostaining experiments were per-formed. Jarid2 (Fig. 2, C and F) and SETDB1 (D and G) areexpressed in the endocardium as well as the myocardium inWT E17.5 hearts.TheDBDof Jarid2 Interacts with the SETDomain of SETDB1—
To investigate the regions involved in the protein-protein inter-action between Jarid2 and SETDB1, GST pull-down assayswere performed (Fig. 3A andB). Various 35S-labeled Jarid2 pro-teins (Fig. 3A, lanes 1–4) were incubated with GST (lanes 5–8)or GST-SETDB1;SET domain only (lanes 9–12) coupled toagarose beads. Both the Jarid2 Ct (Fig. 3A, lane 9) and Jarid2DBD (lane 11) were able to bind theGST-SETDB1;SETdomainonly but not to theGST alone. To perform the reciprocal exper-iment (Fig. 3B), 35S-labeled Jarid2 Ct was incubated with GST(Fig. 3B, lane 1) or various GST-SETDB1 proteins (lanes 2–4)coupled to agarose beads. Our data show that GST-SETDB1 FLand GST-SETDB1;SET only associated with Jarid2 Ct. Theseresults indicate that the DBD of Jarid2 interacts with the SETdomain of SETDB1 in vitro, which is consistent with the resultsof yeast two-hybrid screening.Jarid2 Recruits SETDB1 to the Notch1 Locus—We have
shown that Jarid2 is required for accumulation of H3K9me2andH3K9me3 at the�1150bpNotch1 locus. However, becauseJarid2 is likely enzymatically inactive (18–20), we sought to
FIGURE 2. Jarid2 physically interacts with SETDB1. A, diagram depictingthe protein structures of Jarid2 and SETDB1. Jarid2 contains a JmjN, AT-richinteracting domain (ARID), JmjC, and zinc finger (ZF) domain. SETDB1 con-tains a Tudor, Methyl CpG (MBD) binding domain and a bifurcated Setdomain. A yeast two-hybrid using Jarid2 as bait identified cDNA encoding aa979 –1307 of SETDB1 as the region mediating physical interaction. B, coim-munoprecipitation was performed on E17.5 heart extracts. Lanes 1–3 indicateinput, immunoprecipitate (IP) with non-specific IgG or Jarid2 antibody,respectively, followed by immunoblotting (IB) with SETDB1 antibody. Lanes4 – 6 indicate input, IP with IgG or SETDB1 antibody, respectively, followed byIB with Jarid2 antibody. C–H, immunostaining was performed on E17.5 sec-tions. Brown deposits indicate expression of Jarid2 (C and F) or SETDB1 (D andG). No brown deposits were detected when non-specific IgG was used (E andH). Arrows indicate endocardial cells, and arrowheads indicate myocardialcells. Scale bars � 100 �m (C and F). RV, right ventricle; VS, ventricular septum;LV, left ventricle.
FIGURE 3. GST pull-down assays determine domains mediating interac-tion between Jarid2 and SETDB1. A, various [35S]methionine-labeled Jarid2mutant proteins (lanes 1– 4) were incubated with GST alone (lanes 5– 8) orwith 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-SETonly bound to Jarid2 Ct. �, binding; -, no binding.
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investigate how Jarid2 regulates histone methyl marks on theNotch1 locus. SETDB1, a histone methylase that specificallyconverts H3K9me1 to H3K9me2 and H3K9me3 (26), wasidentified as a putative physical interactor of Jarid2 (Figs. 2Band 3, A and B). As shown in Fig. 4A, SETDB1 is enriched atthe same �1150 bp Notch1 locus where Jarid2 accumulatesin the WT heart, but enrichment is significantly reduced inthe Jarid2 KO mouse heart. This strongly supports ourmodel (Fig. 4B) that Jarid2 is necessary for the recruitment ofSETDB1 to the Notch1 locus where SETDB1 is required forH3K9me2 and H3K9me3 accumulation, leading to genesilencing. Further, it reveals a novel molecular function fortarget gene regulation by Jarid2.Global Determination of Putative Jarid2 Targets in the Devel-
oping Heart—To determine genes regulated through H3K9methat are putative targets of both Jarid2 and SETDB1, we haveconducted genome-wide searches by ChIP-chip using E17.5mouse hearts. As shown in Fig. 5A, ChIP-chip for Jarid2,SETDB1 and H3K9me3 showed 3898, 2001, and 3319 peaks,respectively with a p value � 10�14. Overlapping the datashowed a subset of 594 genes in common among Jarid2,SETDB1 andH3K9me3. These genes represent putative targetsof Jarid2 in the developing heart, which are regulated in associ-ation with SETDB1 via H3K9me3 accumulation. It also allows
for the identification of a subset of Jarid2 targets that are regu-lated in a SETDB1 independent manner. We have previouslyperformed microarray analyses on E17.5 WT versus Jarid2 KOhearts to identify dysregulated genes and molecular pathways(5). When the 594 ChIP-chip genes are overlapped with themicroarray data set, 172 genes are represented with a change ofgreater than 1.2-fold in themutant heart (Fig. 5B). Of these, 107are up-regulated, whereas only 65 are down-regulated.Next, we further define potential targets that are repressed
by Jarid2. Additional analysis by comparing this subset ofup-regulated genes to those contained in the top 11 signifi-cantly up-regulated pathways in Jarid2 KO hearts bymicroarray at E17.5 (5) results in only nine genes that arepotentially repressed by Jarid2 through H3K9me3 bySETDB1 (Fig. 5D). These genes are involved in Notch signal-ing, vasculature development and morphogenesis, regula-tion of cell proliferation and death, cellular adhesion, andheart development. Notch1 is represented in four of thesefive processes, including all of the pathways that couldaccount for the phenotypic defects observed in Jarid2 KOhearts, strongly supporting Notch1 as an endogenous targetof Jarid2 that is critical for cardiac development.
FIGURE 4. Jarid2 is required for SETDB1 accumulation at the �1150 bpregion of the Notch1 locus. A, Jarid2 KO mice have significantly decreasedaccumulation of SETDB1 at the Notch1 locus. Quantitative ChIP was per-formed on E17.5 WT (gray bar) or Jarid2 KO (black bar) mouse hearts using aSETDB1-specific antibody. B, proposed model of Jarid2 regulation of Notch1expression. The expression of Jarid2 is required for the recruitment of SETDB1to the �1150 bp region of the Notch1 locus, resulting in methylation (indi-cated by the asterisk) of H3K9 and Notch1 silencing. Deletion of Jarid2 resultsin 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 ofgenome-wide occupancy of Jarid2, SETDB1, and H3K9me3 by ChIP-chip. B,graph showing the number of genes dysregulated more than 1.2-fold whenthe ChIP-chip was overlapped with the microarray. C, representative Signal-Map peak display for a gene occupied (Notch1) or unoccupied (Lztfl1, Leucinezipper transcription factor-like 1) by Jarid2, SETDB1, and H3K9me3. D, table ofup-regulated ChIP-chip genes that overlap with up-regulated biological pro-cesses identified by microarray analyses. PNPLA6, patatin-like phospholipasedomain containing 6; GRN, granulin, SNED1, Sushi, nidogen, and EGF-likedomains 1; CLDN7, Claudin 7; ROR2, receptor tyrosine kinase-like orphanreceptor 2, ITGA11, integrin � 11; CLSTN1, Calsyntenin 1; NPPB, natriureticpeptide type B.
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Wehave previously shown thatNotch1 expression is elevatedin Jarid2 KO or Jarid2en mouse hearts at later stages of devel-opment, which in part causes cardiac developmental defectsincluding hypertrabeculation and a thin ventricular myocar-dium.Moreover, Jarid2 occupied a specific conserved region ofthe Notch1 genomic locus (5). Although recent work has dem-onstrated that Jarid2 interacts with members of the PRC tomodulate methylation of H3K27 in embryonic stem cells,induced pluripotent stem cells, and epidermal stem cells (20–25), the molecular basis of Notch1 regulation by Jarid2 in thedeveloping heart remains unknown. Therefore, we set out todetermine the precisemolecular consequence of Jarid2 bindingto the Notch1 locus in the developing heart. Here, we demon-strate a novel mechanismwhereby the proper epigenetic meth-ylation of H3K9 by Jarid2 is required for properNotch1 regula-tion in the developing heart. Although it has been reported thatoverexpression of Jarid2 results in recruitment of the histonemethylases G9a and GLP and accumulation of H3K9me1 andH3K9me2 at the CyclinD1 promoter (16), G9a and GLP do notcatalyze H3K9me2 to H3K9me3 and are therefore unlikely tobe involved in Notch1 regulation via H3K9 trimethylation. Inaddition, these results were obtained using an overexpressionsystem in cultured fibroblast cells, which may not reflect phys-iologically relevant mechanisms. Our studies identifiedSETDB1 as a direct binding partner of Jarid2 and demonstratedthat in the developing heart tissue, Jarid2 is essential for therecruitment of SETDB1 to the Notch1 locus where trimethyla-tion takes place, resulting in gene silencing. The decreased lev-els of H3K9me2 and H3K9me3 exhibited in Jarid2 KOmice atlater stages of development likely account for the persistentNotch1 expression.
We also determined global genomic targets for Jarid2,SETDB1, and H3K9me3 by performing ChIP-chip analyses onembryonic hearts. By overlapping all three data sets, we haveidentified 594 putative target genes that are regulated at theepigenetic level by the samemechanisms asNotch1, whichwar-rants further investigation into other potential targets regu-lated by Jarid2 and SETDB1. This represents 15% of all Jarid2targets identified by ChIP-chip, suggesting that Jarid2 criticallyregulates a subset of genes through SETDB1 recruitment andH3K9 trimethylation at later stages of the developing heart. It ispossible that Jarid2 may function through PRC recruitment inthe heart, as demonstrated in stem cells (20–25) or interactwith yet unidentified factors that are involved in epigenetic reg-ulation of other genes.Further examination of the 594 genes identified as putative
targets revealed that 172 (33%) are differentially expressedgreater than 1.2-fold in the hearts of Jarid2KO versusWTmiceat E17.5 by microarray analyses (5). Further, 62% (107) of thesegenes are up-regulated more than 1.2-fold, whereas only 38%(65) are down-regulated more than 1.2-fold. This is consistentwith Jarid2 functioning primarily as a transcriptional repressor.However, it is plausible that Jarid2 may in some cases functionas an activator of transcription. This data set represents a pow-erful and manageable database for identifying putative targetsregulated by Jarid2 through recruitment of SETDB1 and
trimethylation of H3K9. It should be noted that the Notch1gene is among nine putative target genes that are occupied byJarid2, SETDB1, and H3K9me3, and up-regulated more than1.2-fold by microarray.Notch1 is also highly represented in the11 most significantly up-regulated biological processes bymicroarray, strongly correlating it as a bona fide target of Jarid2regulated through H3K9me3 by SETDB1. We cannot rule outthe possibility that Jarid2 regulates other genes by differentmechanisms in the developing heart, including H3K27 methy-lation. Altogether we provide strong evidence for determiningendogenous targets of Jarid2 in the developing heart by employ-ing such combinatorial approaches.It is intriguing that Jarid2 interacts with a subset of proteins
that are involved in histone modifications. In addition to theinteraction with the SET domain containing proteins of thePRC, we have shown that Jarid2 interacts with Zkscan17 (15),which interacts with the SET domain containing histonemeth-ylase NSD1 (21). Additionally, Jarid2 interacts with the SETdomain containing histone methylases GLP and G9a (16). Inour current study, we identified SETDB1 as a direct bindingpartner of Jarid2. Therefore, the involvement of Jarid2 withvarious SET domain containing proteins or complexes raisesthe interesting possibility that Jarid2 acts as a “pan-SETdomain” interacting protein. Jarid2 may regulate many differ-ent cellularmechanisms that are dependent on the specific SETcofactor, which warrants further investigation to reveal newmechanisms of epigenetic regulation involving Jarid2.Here we show a novel mechanism by which Jarid2 silences
Notch1 in the late stages of embryonic cardiac developmentthrough recruiting SETDB1 to facilitate enrichment ofH3K9me2 and H3K9me3 at that locus. This mechanisticinsight coupled with the identification of a subset of genes reg-ulated in a similar process using ChIP-chip and microarrayexperiments allows for a greater understanding of the molecu-lar processes regulated by Jarid2 in the later stages of embryoniccardiac development and other likely developmental processes.
Acknowledgements—We thank Drs. Emery Bresnick and Kirby John-son for valuable discussions and technical assistance.
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Regulation of Notch1 by Jarid2
JANUARY 6, 2012 • VOLUME 287 • NUMBER 2 JOURNAL OF BIOLOGICAL CHEMISTRY 1241
