Changes in the Distributions and Dynamics of Polycomb Repressive Complexes during Embryonic Stem Cell Differentiation

Abstract

Polycomb group (PcG) transcription regulatory proteins maintain cell identity by sustained repression of numerous genes. The differentiation of embryonic stem (ES) cells induces a genome-wide shift in PcG target gene expression. We investigated the effects of differentiation and protein interactions on CBX family PcG protein localization and dynamics by using fluorescence imaging. In mouse ES cells, different CBX proteins exhibited distinct distributions and mobilities. Most CBX proteins were enriched in foci known as Polycomb bodies. Focus formation did not affect CBX protein mobilities, and the foci dispersed during ES cell differentiation. The mobilities of CBX proteins increased upon the induction of differentiation and decreased as differentiation progressed. The deletion of the chromobox, which mediates interactions with RING1B, prevented the immobilization of CBX proteins. In contrast, the deletion of the chromodomain, which can bind trimethylated lysine 27 of histone H3, had little effect on CBX protein dynamics. The distributions and mobilities of most CBX proteins corresponded to those of CBX-RING1B complexes detected by using bimolecular fluorescence complementation analysis. Epigenetic reprogramming during ES cell differentiation is therefore associated with global changes in the subnuclear distributions and dynamics of CBX protein complexes.During differentiation, the pluripotency of embryonic stem (ES) cells is restricted by epigenetic changes (7, 11). Polycomb group (PcG) proteins contribute to the stable inheritance of both pluripotent and differentiated cell states (48, 50). These functions implicate PcG proteins in the control of the transition between pluripotency and differentiation. Genome-wide studies of PcG protein binding in mammalian cells have identified hundreds of genes that bind PcG proteins (8,9,31). The expression of many of these genes is altered during ES cell differentiation.Biochemical studies of PcG proteins have identified two Polycomb repressive complexes (PRCs), PRC1 and PRC2 (12,28,52). PRC2 has lysine methyltransferase activity for K27 of histone H3; PRC1 contains a subunit (Pc in Drosophila and CBX family proteins in mammals) that can bind trimethyl-K27 H3 in vitro (6, 12). Many genes that are bound by PRC1 are enriched in H3 K27 trimethylation (8, 9). These observations, in combination with epistatic relationships among mutations in Drosophila PcG genes (59), have given rise to the model that histone H3 K27 trimethylation by PRC2 is required for the recruitment of the PRC1 complex to specific genes. These results have also been interpreted to indicate that PRC2 initiates silencing and that PRC1 maintains the silenced state.Genetic studies of mice indicate that the functions of PcG proteins are at least in part nonoverlapping since the ablation of genes encoding different PcG proteins produces distinct phenotypes (2,14,17,25,32,38,39,49,(54)(55)(56)(57). Null mutations in the EED and Suz12 subunits of PRC2 eliminate histone H3 K27 trimethylation, but do not prevent the r...

Different polycomb group CBX family proteins associate with distinct regions of chromatin using nonhomologous protein sequences

“…This interpretation is consistent with the observation that a large fraction of CBX proteins is highly mobile in undifferentiated ES cells and in Drosophila embryos (26,28). The CBX proteins that are not chromatin associated could be recruited to new target genes during differentiation.…”

“…In ES cells that stably produced CBX2-H3.2 BiFC complexes (see below), the CBX2 fusions bound to genes recognized by endogenous CBX2 (Fig. S3) and CBX proteins fused to Venus bind selectively to genes that are recognized by endogenous PcG proteins in ES cells (26). CBX4 and CBX6 fused to the GAL4 DNA binding domain repressed an integrated reporter gene carrying GAL4 binding sites (Fig.…”

Ligand-dependent Corepressor LCoR Is an Attenuator of Progesterone-regulated Gene Expression

“…PRC1 complexes form large foci containing numerous factors, including BMI1 polycomb ring finger oncogene (BMI1), visible as discrete nuclear structures (37). Indeed, we found that BMI1 and LCoR coimmunoprecipitated and colocalized in nuclear bodies (supplemental Fig.…”

Mechanisms of transcriptional repression by histone lysine methylation

“…Instead, binding of PRC1 proteins in vivo is highly dynamic (Ficz et al, 2005). Live imaging studies suggest that mobility of PRC1 proteins increases upon induction of ES cell differentiation but decreases again as differentiation progresses (Ren et al, 2008). Our understanding of how PcG proteins mediate their repressive function in vivo is still very limited.…”