Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

Abstract

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.

An Unexpected Regulatory Cascade Governs a Core Function of the Drosophila PRC1 Chromatin Protein Su(z)2

“…The C-terminal regions (CTRs) of Su(z)2 and Psc have been considered the core of these proteins as they are the main contributors of PRC1-mediated silencing; they can, on their own, recapitulate the inhibition of chromatin remodeling, compaction of chromatin, and transcriptional repression exhibited by PRC1 in vitro (King et al 2002(King et al , 2005Francis et al 2004;Lo et al 2009;Beh et al 2012). This is consistent with the fact that, while the mammalian orthologs do not have the CTR, the function of the CTR has been retained in mammals in a stretch of positively charged residues found in the Pc subunit of the PRC1 (Grau et al 2011). Thus, it is believed that the HR serves as the hub of protein-protein interactions which, in part, help direct the CTR to genomic regions requiring regulation by PcG proteins.…”

“…Our findings may also explain the absence of a CTR among the mammalian homologs of Su(z)2, Bmi-1, and Mel-18 (Brunk et al 1991;van Lohuizen et al 1991;Grau et al 2011;Beh et al 2012), as it is possible that mammalian HRs have evolved in such a way as to compensate for the absence of a CTR. On the other hand, it may be that the CTR function of chromatin compaction, first discovered with Drosophila proteins in vitro (Francis et al 2004), still exists in mammals but is found within a different PcG member, M33/ Cbx2 (Grau et al 2011).…”

“…Indeed, the CTR of Psc is necessary and sufficient to bind cyclin B as part of a complex that also requires the HR (Mohd-Sarip et al 2012). Alternatively, the propensity of the CTR and other proteins, such as M33/Cbx2, to bring nucleosomes together in vitro (Francis et al 2004;Lo and Francis 2010;Grau et al 2011) may actually reflect a novel function in vivo, such as a sampling and "reading" of the chromatin landscape and/or the associated chromatin-bound factors, providing an opportunity for the local regulation and prioritization of the varied PcG interactions that occur with the HR (Figure 4D) (Gao et al 2012;Junco et al 2013;Kassis and Brown 2013;Morey et al 2013). Overall, our genetic analysis reveals a novel interaction between the CTR and HR and further study should elucidate the impact of this intramolecular regulation of Su(z)2 on PcG function.…”

“…One set of PcG proteins forms the Polycomb Repressive Complex 1 (PRC1), which can compact chromatin in vitro into a state that is refractory to remodeling and transcription (King et al 2002(King et al , 2005Francis et al 2004;Grau et al 2011;Beh et al 2012;Simon and Kingston 2013). PcG proteins are also believed to effect silencing via the post-translational modification of histones, such as the methylation of histone H3 on K27 as well as both the ubiquitylation and deubiquitylation of histone H2A on K118/9 (Cao et al 2002;Wang et al 2004;Lagarou et al 2008;Scheuermann et al 2010;Schwartz and Pirrotta.…”

Multiple layers of complexity in cis‐regulatory regions of developmental genes

“…Proteins of the Polycomb group, regulate and interact with epigenetic marks, controlling gene expression programs through cell divisions (Schwartz and Pirrotta, 2008). It has been shown that Polycomb-repressive complexes mediate chromatin compaction in flies and mice (Grau et al, 2011). This largescale chromatin change represses the expression of many developmental genes (Isono et al, 2005;Boyer et al, 2006;Lee et al, 2006;Lanzuolo et al, 2007;Sing et al, 2009).…”