Fine-Tuning Silencing

Abstract

Polycomb Repressive Complex 2 (PRC2) modifies chromatin to silence many embryonic patterning genes, restricting their expression to the appropriate cell populations. Two reports in Cell by Peng et al. (2009) and Shen et al. (2009) identify Jarid2/Jumonji, a new component of PRC2, which inhibits PRC2 enzymatic activity to fine-tune silencing.

Covalent modification of histones is central in regulation of gene expression in metazoans. The Polycomb Repressive Complex 2 (PRC2), which trimethylates histone H3 on lysine 27 (H3K27me3), is necessary for the correct spatio-temporal maintenance of silencing at genes involved in developmental patterning, morphogenesis, and organogenesis. The core subunits of mammalian PRC2—enhancer of zeste (Ezh2), the lysine methyltransferase (KMT), and embryonic ectoderm development (Eed) and suppressor of zeste 12 (Suz12), associated proteins that are essential for its enzymatic activity—exist in multiple complexes (Simon and Kingston, 2009). However, the composition and function of the different PRC2 complexes have yet to be fully elucidated. In a recent issue of Cell, papers from the Wysocka and Orkin labs provide a surprising new insight into one PRC2 complex (Peng et al., 2009; Shen et al., 2009). Both groups show that a fraction of PRC2 associates with Jarid2/Jumonji (Jarid2), which inhibits PRC2's KMT and silencing activities. They suggest that Jarid2 fine-tunes the degree of Polycomb-mediated silencing and thereby can influence cell-fate decisions.

Peng et al. and Shen et al. affinity-purified PRC2 from mouse embryonic stem cells (ESCs) by employing tagged core subunits and found that PRC2 associates with Jarid2 (Peng et al., 2009; Shen et al., 2009). In addition, genome-wide localization analyses showed that Jarid2 is found at the vast majority of sites bound by PRC2 in ESCs. Together, these data suggest that a significant fraction of PRC2 is associated with Jarid2 in ESCs.

Jarid2 is the founding member of the Jumonji C (Jmjc) domain family. Jmjc domains are characteristic of proteins that remove methyl groups, the lysine demethylases (KDMs) (Lan et al., 2008). Jarid2 is unusual among Jmjc proteins in that it lacks crucial residues for cofactor binding and is a catalytically inactive KDM (Lan et al., 2008; Shen et al., 2009). The association of KMT and KDM complexes is thought to be important in maintaining chromatin in an on or off state. For example, KMTs that modify histone H3 on lysine 4 (H3K4me), a mark associated with gene activation, associate with the H3K27 KDM, thereby coupling the addition of an activating mark with the removal of a silencing mark (Swigut and Wysocka, 2007). Similarly, Jarid1a, a H3K4 KDM, is reported to associate with PRC2 (Pasini et al., 2008). However, Jarid2 is substantially more abundant than Jarid1a in ESCs and PRC2-Jarid2 is the predominant KMT-Jmjc complex in ESCs. It may be that the association of PRC2 with Jarid2 is one of the reasons that many of the genes silenced by PRC2 in ESCs contain both H3K27me3 and H3K4me3, a combination of silencing and active marks that is thought to be central in keeping these genes poised for expression in the future (Bernstein et al., 2006).

Peng et al. and Shen et al. provide several lines of evidence to indicate that Jarid2 functions as more than just a catalytically inactive KDM. First, Jarid2 is necessary for normal levels of PRC2 association with its target genes. This requirement is mutual, given that Jarid2 binding was also decreased in PRC2 mutant cells. Second, Jarid2 inhibits PRC2 H3K27 KMT activity in vitro. Third, Jarid2 mutant ESCs exhibit increased H3K27 me3 levels at PRC2 targets, despite decreased occupancy of PRC2. Furthermore, whereas PRC2-bound genes are upregulated in PRC2 mutant ESCs, these genes are downregulated in Jarid2 mutant ESCs. Finally, whereas gene expression changes in differentiating PRC2 mutant ESCs are indicative of accelerated differentiation, differentiating Jarid2 mutant ESCs exhibit gene expression changes consistent with delayed differentiation. A similar trend was observed in vivo, when the Xenopus Jarid2 homolog was depleted during frog early embryogenesis. Thus, Jarid2 promotes association of PRC2 with its target genes while simultaneously inhibiting PRC2 enzymatic activity and attenuating silencing, which in turn promotes expression of differentiation-specific genes. The precise mechanism by which Jarid2 inhibits PRC2 activity is not known. However, Jarid2 directly interacts with Suz12 in vitro (Peng et al., 2009), suggesting that it may interfere with the function of this essential PRC2 subunit.

The PRC2-Jarid2 complex is not likely to be the only flavor of PRC2 complex. Jarid2 and PRC2 cofractionate in high-molecular-weight complexes on glycerol gradients; however, there were lower-molecular-weight PRC2 complexes that lacked Jarid2 (Peng et al., 2009). Shen et al. also show that PRC2 interacts with metal response element-binding transcription factor (Mtf2) (Shen et al., 2009), one of three mammalian homologs of Drosophila Polycomblike (Pcl), which binds fly PRC2 and stimulates its activity (Nekrasov et al., 2007). PRC2-Jarid2 and PRC2-Mtf2 are likely to be distinct complexes because Jarid2 and Mtf2 do not copurify. Why are there so many different PRC2-containing complexes? One possibility is that the degree of silencing must be exquisitely controlled to achieve developmentally correct transitions in gene expression patterns as cells differentiate. If PRC2 activity is very high, as is the case in Jarid2 mutant ESCs, then the strength of any developmental signaling pathways that turn on PRC2-silenced genes may not be sufficient to reliably induce expression of their targets. When PRC2 activity is low, as in Eed or Suz12 mutants, target genes are no longer reliably silenced, and such a result can lead to incorrect execution of developmental programs (Chamberlain et al., 2008; Pasini et al., 2007). Indeed, Eed mutant ESCs are prone to differentiation in culture, suggesting that PRC2 is necessary for ESCs to robustly carry out the selfrenewal program. Thus, one possibility is that the ratio of different PRC2 complexes occupying each target gene may be important in determining whether a signaling pathway will trigger maintenance of or release from silencing, thus allowing cells to silence or upregulate a subset of PRC2 targets in response to particular developmental cues.

The near-perfect overlap between Jarid2 and PRC2 in ESCs argues that it is not simply the presence or absence of Jarid2 that regulates PRC2 target gene activity. However, in addition to Jarid2 and Mtf2 and other Pcl homologs, there are several other sources of variability between PRC2 complexes (Simon and Kingston, 2009). Specifically, there are two related genes encoding H3K27 KMTs, Ezh2 and Ezh1, both of which associate with Eed and Suz12 and there are four isoforms of Eed that differ in their N-termini because of alternative translation-start-site usage. Thus, there is potential for considerable combinatorial complexity of PRC2 complexes. Once the composition and activities of different PRC2 complexes is determined, and the factors that direct these different complexes to their target genes are identified, it may be possible to fully understand how PRC2 is utilized to control so many genes in such a wide variety of developmental contexts.