Manipulation of Long-Range Chromatin Interactions through CTCF Phase Separation for Cell Fate Control. Manipulation of Long-Range Chromatin Interactions through CTCF Phase Separation for Cell Fate Control

Gene expression is controlled under spatial chromatin structures with short-range in topologically associating domains (TAD) and long-range chromatin interactions between TADs, compartments or chromosomes, and disruption of chromatin structure leads to human diseases. The mechanism of short-range chromatin interactions has been well characterized by loop-extrusion model, but little is known about how long-range chromatin interactions are organized. Here, we demonstrate that CTCF contributes to long-range chromatin interactions via phase separation. Surprisingly, RYBP is required for the phase separation and long-range chromatin organization of CTCF. Artificial CTCF phase seperation restores the long-range chromatin interactions and corresponding gene expression which were eliminated by RYBP depletion, and manipulation of CTCF phase separation also maintains pluripotency and inhibits differentation of embryonic stem cells. These findings support a model that long-range chromatin interactions are organized through phase sepearation of architectural protein, and further reveals the distinct mechanisms of architectural protein in organizing short-range and long-range chromatin interactions. Overall design: Examination of RNA-seq, Hi-C, CTCF HiChIP, RYBP HiCHIP NGS data before or/and after RYBP knock out.

基因表达受空间染色质结构的调控：这类结构在拓扑关联结构域（topologically associating domains, TAD）内部存在短程染色质相互作用，而在TAD之间、染色质区室之间乃至不同染色体之间则存在长程染色质相互作用；染色质结构的紊乱可引发人类疾病。短程染色质相互作用的机制已通过环挤出模型得到充分阐释，但长程染色质相互作用的组装机制目前仍知之甚少。本研究证实，CCCTC结合因子（CTCF）可通过相分离（phase separation）参与长程染色质相互作用的调控。令人意外的是，RYBP是CTCF实现相分离以及完成长程染色质组装所必需的蛋白。人工诱导的CTCF相分离可恢复RYBP敲除所消除的长程染色质相互作用及对应基因表达；而调控CTCF的相分离还能够维持胚胎干细胞的多能性，并抑制其分化。上述研究结果支持“长程染色质相互作用通过结构蛋白的相分离进行组装”的模型，并进一步揭示了结构蛋白在调控短程与长程染色质相互作用时所采用的差异化机制。整体实验设计：对RYBP敲除前后的RNA测序（RNA-seq）、Hi-C、CTCF HiChIP、RYBP HiChIP及下一代测序（next-generation sequencing, NGS）数据进行分析。