Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (ChIP-Seq). Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (ChIP-Seq)

Whether RNA G-quadruplexes (rG4) form extensively in vivo and what roles they play remain actively debated. Among their proposed functions is recognition of Polycomb repressive complex 2 (PRC2), but how the interaction results in epigenetic regulation is not understood. Here we demonstrate that rG4s form dynamically during ES cell differentiation and require ATRX’s helicase function to unwind competing secondary structures. Mutating ATRX causes rG4 depletion on a transcriptome-wide basis and dramatically increases gene expression. We identify and mutate rG4s within Xist RNA and mechanistically separate PRC2’s recruitment versus catalysis. Surprisingly, although rG4s recruit PRC2, unfolding the rG4 structure causes PRC2 hyperactivation, entrapment of PRC2 in the S1 chromosomal compartment, and loss of gene silencing. Thus, we link dynamic rG4 folding and unfolding to PRC2 recruitment, trans-compartmental Xist migration, regulated activation of PRC2, and whole-chromosome gene silencing. Overall design: Modified existing rG4-seq and RT-Stop protocols were performed and generated an “in vitro” and “in vivo” pipeline, based on differential sensitivity of various RNA structures to dimethyl sulfate (DMS), reverse transcriptase (RT), and the monovalent cations Li+ versus K+.

RNA G-四链体（RNA G-quadruplexes，rG4）是否在体内广泛形成以及其发挥何种功能，目前仍存在广泛的学术争议。其已被提出的功能之一是识别多梳抑制复合体2（Polycomb repressive complex 2，PRC2），但该相互作用如何介导表观遗传调控，目前尚未阐明。本研究证实，rG4在胚胎干细胞（ES）分化过程中发生动态形成，且需要ATRX的解旋酶功能来解开竞争性RNA二级结构。突变ATRX会导致转录组水平的rG4耗竭，并显著上调基因表达水平。我们在Xist RNA中鉴定并突变了rG4位点，从机制上区分了PRC2的招募功能与催化活性。令人意外的是，尽管rG4能够招募PRC2，但解开rG4结构会导致PRC2过度激活、PRC2滞留于S1染色体区室，并引发基因沉默功能丧失。综上，本研究将动态的rG4折叠与解折叠过程，与PRC2招募、跨区室Xist迁移、PRC2的调控激活以及整条染色体的基因沉默建立了关联。实验设计：本研究对已有的rG4测序（rG4-seq）与逆转录终止（RT-Stop）实验方案进行了优化，基于不同RNA结构对硫酸二甲酯（dimethyl sulfate，DMS）、逆转录酶（reverse transcriptase，RT）以及锂离子（Li+）与钾离子（K+）的差异敏感性，构建了体外（in vitro）与体内（in vivo）两套实验流程。