JMJ interacts with PRC2
To identify proteins associated with PRC2, we affinity-purified the complex using a previously described in vivo
biotinylation strategy (Wang et al., 2006
). Nuclear extracts from ESCs coexpressing bacterial biotin ligase (BirA) and subendogenous levels of b
iotin- and F
LAG-tagged EED, EZH1 or EZH2 referred to as bfEED, bfEZH1, bfEZH2 (Shen et al., 2008
) were subjected to streptavidin-mediated coimmunoprecipitation or tandem coimmunoprecipitation mediated by anti-FLAG antibody and streptavidin. Interacting proteins were then identified by mass spectrometry sequencing (; Table S1
JMJ interacts with the PRC2 complex
In addition to known PRC2 core components including EZH1/EZH2, EED, SUZ12, RBBP4 and AEBP2, we identified several novel interacting partners of PRC2 including JUMONJI (JMJ), metal response element-binding transcription factor 2 (MTF2) and serine/threonine kinase 38 (STK38). To confirm the putative interacting proteins and to expand the PRC2 interaction network, we then tagged MTF2 and JMJ with a biotin tag and FLAG epitope (herein referred to as bfMTF2 and bfJMJ) in ESCs. The reverse tagging analysis confirmed association of PRC2 with MTF2 and JMJ (Table S5
). We did not detect peptides representing the H3K9 methyltransferases G9a and GLP that were reported to interact with JMJ when overexpressed in NIH3T3 cells (Shirato et al., 2009
Based on proteomic results, we constructed an "interactome" surrounding PRC2 (), which includes the core PRC2 components, MTF2, JMJ, STK38, RBBP7 and additional JMJ interacting proteins that may indirectly or transiently associate with PRC2. Interestingly, bfJMJ pulled down few peptides representing MTF2, and bfMTF2 did not capture JMJ (Table S1
). Thus, these two PRC2-associated proteins may not coexist in the same PRC2 complex. To validate the above interactions, we co-expressed V5-tagged MTF2, HA-tagged JMJ, bfEED and bfEZH2 in 293T cells. Coimmunoprecipitation of MTF2, JMJ or EED / EZH2 pulled down the other three components (), suggesting their coexistence in one protein complex. To verify binary interactions, we coexpressed V5-tagged MTF2 or HA-tagged JMJ with one of bfEED, bfEZH1 and bfEZH2 in 293T cells. Both MTF2 and JMJ bind to EED, EZH1 or EZH2 ().
MTF2 (also called PCL2) is a member of the Polycomblike / PHF1 (PCL1) family proteins, which associate with PRC2 in fly and in mammal (Cao et al., 2008
; Nekrasov et al., 2007
; Sarma et al., 2008
; Savla et al., 2008
). Because of the recognized importance of Jumonji family proteins in regulating chromatin structure, cellular function and development (Cloos et al., 2008
; Nottke et al., 2009
), we focused on the interaction of JMJ with PRC2. Fractionation of nuclear extracts from CJ7 ESCs by gel filtration shows that JMJ comigrates with EZH2 with two peaks centered at 3908 kD and 395 kD (), indicating that JMJ is possibly associated with a fraction of PRC2 in ESCs. Streptavidin capture of bfEZH2 and bfEED in ESCs precipitates endogenous JMJ (). An anti-SUZ12 antibody mediates coimmunoprecipitation of JMJ but not RBP2, the H3K4 demethylase, in wild-type ESCs ( and S1
). Reciprocal coimmunoprecipitation mediated by an anti-JMJ antibody captures endogenous EZH2, EED and SUZ12 (). These results demonstrate that JMJ interacts with PRC2 in a physiological setting.
mutant ESCs JMJ fails to coimmunoprecipitate with EED, and captures significantly less SUZ12 compared to that in wild-type cells (). In addition, an EZH2 mutant lacking the catalytic SET domain (bfEZH2dSET) (Shen et al., 2008
) coimmunoprecipitates with SUZ12 but not JMJ (). These results implicate EZH2 and its SET domain in mediating in vivo
association of JMJ and PRC2. EED appears dispensable for this interaction as JMJ coimmunoprecipitates with EZH2 and SUZ12 in Eed−/−
ESCs. Endogenous JMJ pulls down PRC2 not only in ESCs, but also in mouse erythroleukemia (MEL) cells and F9 embryonic carcinoma cells (). Therefore, the interaction between JMJ and PRC2 is not unique to pluripotent ESCs.
JMJ colocalizes with PRC2 on chromatin
JMJ contains a conserved ARID / BRIGHT domain, which can bind DNA (Kim et al., 2003
). We sought to identify DNA targets of JMJ. Chromatin immunoprecipitation (ChIP) in ESCs using antibody to JMJ shows that endogenous JMJ is significantly enriched at promoters of PRC2 target genes (). To identify gene targets of JMJ in a global fashion, we performed ChIP-chip on Affymetrix promoter tiling arrays. Out of 644 target genes bound by JMJ (; Table S7
), 632 (98%) genes overlap with genes marked by H3K27me3 (Shen et al., 2008
). Moreover, 81% ~ 98% of JMJ target genes overlap with targets of EZH2, SUZ12 and bfEED (; Table S7A
). The extent of overlap between JMJ and PRC2 / H3K27me3 targets greatly exceeds the reported colocalization between RBP2 and H3K27me3, in which only 195 out of 606 RBP2 target genes overlap with H3K27me3 targets (Pasini et al., 2008
JMJ colocalizes with the PRC2 and H3K27me3 mark on chromatin
Results with the conventional ChIP method are highly dependent on the quality of available antibodies to the target protein, whereas bioChIP obviates the need for antibodies and takes advantage of the strong interaction between biotin and streptavidin (Kim et al., 2008
; Shen et al., 2008
). To refine the genome-wide targets of JMJ, we analyzed ESCs expressing subendogenous level of bfJMJ (data not shown) and confirmed the enrichment of bfJMJ on PRC2 targets (). BioChIP-chip of bfJMJ on Affymetrix whole genome tiling arrays revealed many more targets (3695 genes) than JMJ antibody-mediated ChIP-chip ( and Table S9
). As 97% of JMJ ChIP-chip targets overlap bioChIP-chip targets (Table S7
), bioChIP-chip faithfully maps comprehensive coverage of chromatin binding of endogenous JMJ. Over 80% of H3K27me3, bfEZH1 or bfEZH2 targets overlap those of bfJMJ (Table S7
), confirming genome-wide colocalization of JMJ with PRC2 and H3K27me3. JMJ occupies genomic regions in close proximity to transcription start sites (TSS) (). Similar to H3K27me3 target genes, JMJ target genes are enriched in development-related functions (). Thus, JMJ interacts with PRC2 at both protein and chromatin levels.
JMJ-containing protein complex shows methyltransferase activity on H3K27
To address functional implication of the interaction of JMJ with PRC2, we assayed JMJ-associated proteins for lysine methyltransferase (KMT) activity. Indeed, proteins that coprecipitate with bfJMJ in ESCs demonstrate KMT activity towards core histones in a dose-dependent manner similar to the bfEZH2-containing complex captured by streptavidin (). In addition, the endogenous JMJ-containing complex precipitated by an anti-JMJ antibody shows KMT activity towards nucleosomes, which are the natural substrates in vivo ().
Histone methyltransferase assays show intrinsic KMT activity within the JMJ-containing complex
To study substrate specificity, recombinant histones bearing methyl lysine analogues (MLAs) were used to examine desired methylation patterns on histone H3. These MLAs (indicated as Kc) behave similarly to their natural counterparts (Simon et al., 2007
). The bfEZH2-containing complex shows decreasing activity on the H3 substrates with increasing numbers of methyl groups on residue 27 (). This result is consistent with the report that mono-methylation activity of PRC2 is dominant to its tri-methylation activities in vitro
(Sarma et al., 2008
). A weak methylation signal observed on H3Kc27me3, which contains saturated methyl groups, may reflect non-specific activity of PRC2 on lysine 9 in vitro
. Although PRC2 specifically acts on H3K27 in vivo
, recombinant human PRC2 exhibits weak KMT activity on H3K9 in vitro
(Kuzmichev et al., 2002
The bfJMJ complex exhibits similar substrate specificity as the bfEZH2 complex with the highest and lowest activity towards unmethylated H3 and H3Kc27me3, respectively, supporting physical existence of JMJ and PRC2 in one protein complex. Moreover, the endogenous JMJ-containing complex shows similar activity on unmethylated H3 and H3Kc9me3 substrates, but significantly weaker activity on the H3Kc27me3 substrate (), implying that the JMJ-PRC2 complex specifically acts on H3K27, but not H3K9.
JMJ inhibits PRC2 activity in vitro
Mammalian JMJ contains several conserved motifs including JmjN, ARID/BRIGHT, JmjC and C5HC2 zinc finger domains (Figure S2
). Mutation of the critical residues involved in cofactor binding in the JmjC domain predicts that JMJ is inactive as a demethylase, a function distinguishing many Jumonji family members (Cloos et al., 2008
). Consistent with the prediction, we failed to detect lysine demethylase (KDM) activity of JMJ regardless of the presence of EZH2 (Figure S3
). Moreover, addition of JMJ had no obvious effect on known KDMs (Figure S3
To ask whether JMJ assembles as a stoichiometric component of PRC2, we reconstituted JMJPRC2 through baculoviral expression. Anti-HA coimmunoprecipitation targeted to EED captures JMJ, which is present at one-fourth the amount of EZH2. Therefore, JMJ is a substoichiometric component of PRC2 (). This result suggests that PRC2 may exist in two forms: JMJ-containing and JMJ-free PRC2 complexes.
JMJ inhibits methyltransferase activity of PRC2
A chromatin fraction mainly comprised of oligonucleosomes from Hela cells contains endogenous KMT activity (). Addition of increasing amounts of JMJ but not bovine serum albumin (BSA) markedly inhibits this activity. Close interaction between JMJ and PRC2 suggests that JMJ may directly regulate the KMT activity of PRC2. To test this possibility, we reconstituted KMT reactions with recombinant PRC2 and histone H3 MLAs with the defined methylation pattern at residues Kc27 and Kc9.
Addition of JMJ did not affect PRC2-mediated mono-methylation on H3 and H3Kc9me3 substrates (). JMJ dramatically down-modulates PRC2 activity on H3Kc27me1 and me2 substrates in a dose-dependent manner (). BSA has no effect on PRC2, indicating the specificity of the assay. JMJ failed to inhibit PRC2 on chicken core histones (data not shown), presumably due to dominant monomethylation catalyzed by PRC2 in vitro, which may mask the inhibitory effect of JMJ on di- and tri-methylation. The JMJ mutant (dJc) lacking the C-terminal JmjC and C5HC2 domains also inhibits di- and tri-methylation activity of PRC2 (), suggesting that the JmjC and C5HC2 domains are dispensable for JMJ activity on PRC2.
To test whether JMJ specifically inhibits PRC2 activity on more physiological substrates, we assembled recombinant histone octamers and nucleosomes containing H3Kc27me0, me1 or me2. Consistent with histone substrates, JMJ inhibited PRC2 mediated di- and tri-methylation in a dose-dependent manner on MLA octamers and nucleosomes (). JMJ also inhibited monomethylation activity of PRC2 on H3Kc27me0 octamers and nucleosomes, indicating that JMJ-PRC2 may exhibit different enzymatic activities towards H3 histone, octamers, core histones (which contain octamers plus the linker histone H1) and nucleosomal substrates in vitro. Nevertheless, these results demonstrate that JMJ inhibits di- and tri-methylation activity of PRC2.
JMJ fine-tunes the H3K27me3 level in vivo
To investigate how JMJ regulates PRC2 function in vivo
, we isolated Jmj
conditional knockout (fl/fl
) ESCs. Deletion of exon 3 creates a frame shift and subsequent termination mutation (Figure S2A
), resulting in a mutant peptide of 15 amino acids that contains none of the known functional motifs of JMJ (Mysliwiec et al., 2006
). By Cre
recombinase-mediated excision of the fl
alleles in vitro
, we established Jmj−/−
ESCs, in which JMJ expression is completely abolished and JMJ is no longer detected at PRC2 target loci by ChIP (). Anti-SUZ12 coimmunoprecipitation in Jmj−/−
cells fails to detect a protein band representing JMJ (), confirming the specificity of the interaction between JMJ and PRC2. Jmj−/−
ESCs show normal expression of pluripotency markers (Table S14
) and proliferate at a similar rate as wild-type ESCs under standard conditions (data not shown). Thus, JMJ is dispensable for ESC self-renewal and maintenance.
As JMJ inhibits PRC2 tri-methyltransferase activity in vitro
, we investigated whether loss of JMJ affects H3K27me3 levels in vivo
. Global levels of H3K27 methylation, as well as other histone marks, are not affected in Jmj−/−
ESCs (Figure S4
), consistent with a role of JMJ as a modifier of PRC2. However, moderate, but consistent, enhancement of H3K27me3 binding at individual PRC2 target loci is observed in Jmj−/−
To study the effect of Jmj
loss on gene expression of PRC2 and H3K27me3 targets, we performed microarray profiling of Jmj−/−
ESCs (Table S14
). By G
nalysis (GSEA), we ranked genes from high to low based on the correlation between their expression levels in these two cell types (). Genes that are upregulated in Jmj−/−
ESCs compared to Jmjfl/fl
ESCs are ranked at the top of the list, while genes that are downregulated are ranked toward the bottom of the list. We previously identified a subset of H3K27me3 target genes referred to as ‘H3K27me3-(WT)-Day6 UP’ genes that are repressed in undifferentiated ESCs but upregulated at day 6 of ESC differentiation (Shen et al., 2008
). We then asked where members of this H3K27me3 target gene set are distributed in the ranked dataset. We find that H3K27me3 target genes distribute toward the bottom of the rank list with a significant normalized enrichment score (NES) of −2.9 (), indicating reduced expression in Jmj−/−
ESCs. Thus, accentuated repression of H3K27me3 genes is consistent with ChIP analysis, indicating that increased presence of H3K27me3 on chromatin may further repress PRC2 targets that normally exhibit low levels of transcription.
The enrichment of JMJ at PRC2 targets is markedly reduced in Ezh2−/−
ESCs (), indicating a requirement of EZH2 and EED in targeting JMJ to chromatin. This result is consistent with the observation that JMJ lacks specific binding affinity to methylated histones, as revealed by histone peptide binding assays (Figure S5
). On the other hand, enrichment of SUZ12, EZH2 and EED at PRC2 targets is significantly decreased in Jmj−/−
ESCs (), pointing to a positive role of JMJ in facilitating PRC2 binding to chromatin. Thus, JMJ modulates PRC2 function in opposing ways. As PRC2 and UTX / JMJD3 function as lysine methyltransferases (KMTs) and demethylases (KDMs), respectively, to control “on” and “off” switches of H3K27me3, JMJ functions as a modifier of PRC2 to fine-tune the level of H3K27me3 by promoting chromatin binding, while also inhibiting the KMT activity of PRC2 ().
JMJ is required for proper ESC differentiation
The core components of PRC2 including EZH2, EED and SUZ12 appear to be more important in executing than maintaining pluripotency (Pasini et al., 2007
; Shen et al., 2008
). To investigate the role of JMJ in regulating ESC pluripotency, we differentiated Jmj−/−
ESCs by withdrawal of leukemia inhibitory factor (LIF) and performed microarray expression profiling.
Global gene expression in Jmj−/− ESCs was compared to that in Jmjfl/fl ESCs by GSEA. H3K27me3 targets are distributed primarily toward the bottom of the ranked list in their enrichment profiles with the lowest NES of −3.0 at day 4 compared to −2.5 and −2.0 at day 6 and 8 of differentiation, respectively (). Consistent with GSEA which provides a statistical comparison of a prior defined gene set between two cell types, heatmap analysis shows that the activation of H3K27me3 target genes in Jmj−/− ESCs is delayed and not activated to the same extent as in Jmjfl/fl cells during differentiation ().
Jmj is required for ESC differentiation
Upon LIF withdrawal pluripotent ESCs differentiate into three germ layers including neuroectoderm, mesoderm and endoderm. Mesoendoderm (ME) is a transient cell state prior to further differentiation into mesoderm and endoderm. Both JMJ and H3K27me3 occupy a set of genes critical for development. We studied the effect of JMJ loss on lineage differentiation, particularly genes enriched in neuronal progenitors (NP) and mesoendodermal (ME) lineages, which were previously defined as “NP-high” genes and “ME-high” genes, respectively (Shen et al., 2008
GSEA and heatmap analysis indicate significant underrepresentation of NP-high genes in Jmj−/− cells during differentiation (). ME-high genes in Jmj−/− cells are also significantly downregulated at day 4 and day 6 of differentiation, but eventually become overrepresented with a positive NES of 1.7 at day 8 of differentiation (). Thus, loss of JMJ results in compromised and delayed differentiation of ESCs. Imposition of excessive H3K27me3 marks in the absence of JMJ may render a less permissive chromatin conformation on target genes and attenuate rapid reconfiguration of chromatin states in response to differentiation signals.
To confirm microarray analysis, we analyzed expression of marker genes in wild-type (Jmjfl/fl
) and polycomb mutant ESCs including Jmj−/−
cells (). Jmj
transcripts are downregulated 6-fold and 8-fold, respectively, at day 4 and day 8 of differentiation, implying early involvement of JMJ in regulation of ESC differentiation. Upon differentiation higher expression of ESC specific genes, including the pluripotency marker Oct4
, is observed in Jmj−/−
cells as compared to Jmjfl/fl
cells ( and S6
). Delayed attenuation of pluripotency genes confirms a delay in differentiation.
Fgf5 is a marker for primitive ectoderm, a transient cell state that pluripotent ESCs must proceed through before differentiating to three germ layers. In wild-type ESCs, the expression of Fgf5 reaches its peak at day 2 of differentiation and is gradually downregulated thereafter (). This pattern of expression is consistent with a transitory existence of primitive ectoderm during development. However, Ezh2−/− and Eed−/− cells show abnormal activation of Fgf5 at day 2 but an abrupt drop to a significantly lower level at day 4, suggesting rapid entrance into and exit from the primitive ectoderm state. Despite normal activation at day 2 in Jmj−/− cells, Fgf5 expression progressively rises to nearly 8-fold higher than that in wild-type cells at day 6. Persistent, high level expression of Fgf5 suggests that Jmj−/− cells differentiate into primitive ectoderm but further development is stalled at this stage.
Consistent with prolongation of the primitive ectodermal state, Jmj−/− cells show delayed commitment to the three somatic lineages (). For example, neuronal gene Sox11 fails to be upregulated upon differentiation in mutant cells. Brachyury (T) – an early marker for mesoendoderm reaches its peak expression at day 4 in wild-type cells. However, Jmj−/− cells weakly express T at day 4 and show peak activation at day 6, at a time when T expression in wild-type cells has already been downregulated. Additional mesoderm markers, such as Sp5, Bmp4 and Hoxa1, also show delayed and compromised activation. In Ezh2−/− and Eed−/− cells, both T and Sp5 show a similar time-course of activation as wild-type ESCs, but the extent of activation is reduced, reflecting induction but failure to sustain mesoendoderm in these mutant cells.
Expression of CD44, a cell surface marker in mesenchymal stem cells important for epithelial-mesenchymal transition (EMT), is not activated appropriately in Jmj−/− cells (). In contrast, CD44 expression is activated but not sustained in Ezh2−/− and Eed−/− cells. Lastly, cell cycle regulators, including cyclin D2 (Ccnd2) and Cdkn2a (p16), which are highly expressed in all three germ layers, are expressed at abnormally high levels in Ezh2−/− and Eed−/− cells, suggesting that differentiation may be initiated but cannot be sustained due to cell-cycle arrest. In contrast, Ccnd2 and Cdkn2a are not activated until day 8 of differentiation in Jmj−/− cells with expression levels significantly lower that those in Jmjfl/fl cells (). Therefore, global and individual gene analysis demonstrate that Jmj−/− ESCs fail to respond to differentiation signals promptly and exhibit delayed, compromised lineage commitment.