Abstract
Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.
Highlights
[Keywords: polycomb; topologically associating domains (TADs); gene repression; nuclear organization; embryonic stem cells; gene regulation; epigenetics; histone modifications]
DAPI staining of 2D nuclear preparations revealed a significant increase in nuclear area in the absence of Polycomb-repressive complex 1 (PRC1) (Ring1b−/−) when compared with parental Ring1b+/+ mouse embryonic stem cells (mESCs) (Fig. 1A; Supplemental Fig. S1A)
Fluorescence-activated cell sorting (FACS) did not identify an altered cell cycle profile between Ring1b+/+ and Ring1b−/− cells (Supplemental Fig. S1B). This suggested that the increase in nuclear size in Ring1b−/− cells is a direct consequence of PRC1 depletion on nuclear structure, rather than an accumulation of cells in G2
Summary
[Keywords: polycomb; topologically associating domains (TADs); gene repression; nuclear organization; embryonic stem cells; gene regulation; epigenetics; histone modifications]. Loss of these architectural PRC1 subunits leads to the dissolution of nanometre scale “polycomb bodies” containing high local concentrations of polycomb proteins (Isono et al 2013; Wani et al 2016; Plys et al 2019; Tatavosian et al 2019) These data support the idea that CBX and PHC proteins bestow cPRC1 with the capacity to fold chromatin into discrete nuclear domains and suggest a mechanistic role for chromatin interactions and nuclear clustering in PRC1-mediated transcriptional repression. What factors determine which distal PRC1 targets will physically interact? Does PRC1 create a topology that anchors multiple loci simultaneously in a single cell and, if so, do such structures occur in vivo? What is the cause/consequence relationship between chromatin structure and gene derepression in cells lacking RING1B? In this study, we used both Hi-C and DNA Fluorescence in
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