A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Genes & development (2020-05-23)
Shelagh Boyle, Ilya M Flyamer, Iain Williamson, Dipta Sengupta, Wendy A Bickmore, Robert S Illingworth
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.

MATERIALS
Product Number
Brand
Product Description

SAFC
MEM Non-essential Amino Acid Solution (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Sodium pyruvate solution, 100 mM, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
SYBR® Green I nucleic acid gel stain, 10,000 × in DMSO
Sigma-Aldrich
Gelatin from porcine skin, powder, gel strength ~300 g Bloom, Type A, BioReagent, suitable for electrophoresis, suitable for cell culture
Sigma-Aldrich
4-Thiouridine, ≥98%