RBBP4 regulates pluripotent-to-2C-like state transition through modulating heterochromatin assembly (CUT&Tag). RBBP4 regulates pluripotent-to-2C-like state transition through modulating heterochromatin assembly (CUT&Tag)

Cellular totipotency is critical for generating a whole organism, yet how to establish totipotency is still poorly illustrated. Unlike pluripotent stem cells, abundant transposable elements (TEs) are activated in the totipotent cells. Here, we show that histone chaperone RBBP4 but not its homologous RBBP7 is indispensable in maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms pluripotent state to a totipotent-like (also known as 2C-like) state. Mechanistically, RBBP4 could recruit G9a and KAP1 to bind on retrotransposons, especially endogenous retroviruses. RBBP4 degradation reduces the binding of G9a-mediated H3K9me2 on ERVL (particularly MERVL) and KAP1-mediated H3K9me3 on ERV1/ERVK, respectively. Moreover, RBBP4 facilitates nucleosome occupancy through chromatin remodeler CHD4 and RBBP4 depletion leads to attenuation of CHD4 binding and nucleosome occupancy on TEs. Together, our study reveals the important roles of RBBP4 in heterochromatin assembly and its loss activates TEs in mESCs, opening a new way to obtain totipotent cells in vitro. Overall design: Examination the binding sites of H3K9me2 in Rbbp4-AID mES cells with or without auxin-induced degradation of RBBP4.

细胞全能性（cellular totipotency）对于完整有机体的生成至关重要，然而当前学界对其建立的具体机制仍阐释不足。与多能干细胞不同，全能性细胞中会激活大量转座因子（transposable elements, TEs）。本研究发现，组蛋白伴侣RBBP4（而非其同源蛋白RBBP7）对于维持小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的细胞身份不可或缺。通过生长素诱导降解RBBP4（而非RBBP7），可将多能状态重编程为类全能性状态（亦称2C样状态）。从机制层面来看，RBBP4可招募G9a与KAP1结合至逆转录转座子，尤其是内源性逆转录病毒。具体而言，RBBP4降解会分别减弱G9a介导的H3K9me2在ERVL（尤其是MERVL）上的结合，以及KAP1介导的H3K9me3在ERV1/ERVK上的结合。此外，RBBP4可通过染色质重塑因子CHD4促进核小体占据；RBBP4耗竭会削弱CHD4在转座因子上的结合能力与核小体占据水平。综上，本研究揭示了RBBP4在异染色质组装中的关键作用，其缺失会激活小鼠胚胎干细胞中的转座因子，为体外获取全能性细胞提供了全新途径。实验设计概述：检测在生长素诱导RBBP4降解与否的情况下，Rbbp4-AID小鼠胚胎干细胞中H3K9me2的结合位点。