Polycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells

Abstract

SUMMARYPolycomb group (PcG) proteins play an important role in the control of developmental gene expression in higher organisms. In mammalian systems, PcG proteins participate in the control of pluripotency, cell fate, cell cycle regulation, X chromosome inactivation and parental imprinting. In this study we have analysed the function of the mouse PcG protein polycomblike 2 (Pcl2), one of three homologues of the Drosophila Polycomblike (Pcl) protein. We show that Pcl2 is expressed at high levels during early embryogenesis and in embryonic stem (ES) cells. At the biochemical level, Pcl2 interacts with core components of the histone H3K27 methyltransferase complex Polycomb repressive complex 2 (PRC2), to form a distinct substoichiometric biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 facilitates both PRC2 recruitment to the inactive X chromosome in differentiating XX ES cells and PRC2 recruitment to target genes in undifferentiated ES cells. The role of Pcl2 in PRC2 targeting in ES cells is critically dependent on a conserved PHD finger domain, suggesting that Pcl2 might function through the recognition of a specific chromatin configuration. KEY WORDS: ES cell, Polycomb, X inactivation, MousePolycomblike 2 facilitates the recruitment of PRC2 Polycomb group complexes to the inactive X chromosome and to target loci in embryonic stem cells Li et al., 2010; Landeira et al., 2010) but its function is as yet undetermined. Finally, the Polycomblike (Pcl) protein associates with PRC2 in Drosophila (O'Connell et al., 2001;Tie et al., 2003;Papp and Muller, 2006) and in mammalian cells (Cao et al., 2008;Sarma et al., 2008) and has been proposed to have a role in stimulating H3K27me3 activity and/or targeting of the complex (Nekrasov et al., 2007;Cao et al., 2008;Sarma et al., 2008). In this study we have analysed the function of the mouse Pcl2 (Mtf2 -Mouse Genome Informatics) protein, one of three homologues of Pcl found in mammalian cells. We find that Pcl2 is expressed at high levels during early embryogenesis and in ES cells. Pcl2 interacts with the core PRC2 complex to form a stable and distinct biochemical complex, Pcl2-PRC2. Functional analysis using RNAi knockdown demonstrates that Pcl2-PRC2 is important in PRC2 recruitment to the Xi in differentiating XX ES cells and also for PRC2 recruitment to target genes in undifferentiated ES cells. A conserved PHD finger domain in Pcl2 is required for PRC2 targeting in ES cells. MATERIALS AND METHODS Cell lines and embryosGrowth and maintenance of trophoblast stem (TS) and ES cell lines were as described previously (Penny et al., 1996;Mak et al., 2002). ES cell differentiation was achieved by embryoid body formation via removal of LIF from the medium. The MP fibroblast cell line was derived in house from a male PGK mouse.Preimplantation and postimplantation mouse embryos were obtained from timed mating of C57B16ϫCBA F1 animals as described previously (Sheardown et al., 1997). Immunofluorescence (IF) and RNA fluorescent in ...

Polycomb-like proteins link the PRC2 complex to CpG islands

“…In contrast, H3K27me3 was only mildly affected by the level of chromatin-bound MTF2 (Fig. 4e, f), which is similar to the previously observed minor consequences on H3K27me3 levels after MTF2 or PHF19 depletion in vivo 9,10 (Extended Data Fig. 5c).…”

Noncoding RNA and Polycomb recruitment

“…One possibility is that other elements within the Xist transcript also bind directly to core PRC2 components. A further study demonstrated that Polycomblike2 (PCL2), a substoichiometric component of PRC2, is important for PRC2 recruitment by Xist RNA (Casanova et al 2011). However, depletion of PCL2 also affected PRC2 recruitment at target sites other than Xi, suggesting a more general function in PRC2 binding to chromatin.…”

Composition, recruitment and regulation of the PRC2 complex

“…37 Primary sequence analysis reveals the presence of Tudor and PHD domains, which may bind to modified histones. Recent studies 38,39 have shown that the second PHD domain of MTF2 was required for targeting the PRC2 complex in ESCs. Previous studies have also shown that the PHF1 increases activity of the PRC2 complex in somatic cells.…”