G-quadruplex folding in Xist RNA antagonizes PRC2 activity for step-wise regulation of X-chromosome inactivation

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. While PRC2 binds G-tracts with potential to form RNA G-quadruplexes (rG4), whether rG4’s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model, here we identify multiple folded rG4’s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4’s. High-affinity rG4 binding inhibits PRC2’s histone methyltransferase activity and stabilizing rG4 in vivo antagonizes H3K27me3 enrichment on the inactive X-chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding dynamics control PRC2 activity, H3K27me3 enrichment, and the step-wise regulation of chromosome-wide gene silencing.RNA对多梳抑制复合体2（Polycomb repressive complex 2, PRC2）的调控机制仍是一个尚未解决的科学问题。尽管PRC2可结合具备形成RNA G-四链体（RNA G-quadruplexes, rG4）潜力的G串序列，但rG4在体内是否广泛折叠，以及PRC2结合的是折叠态还是未折叠态的rG4，目前仍不得而知。本研究借助X染色体失活模型，在Xist RNA中鉴定出多个折叠态rG4，并证实PRC2偏好结合折叠态的rG4。高亲和力的rG4结合会抑制PRC2的组蛋白甲基转移酶活性，而在体内稳定rG4则会拮抗失活X染色体上的H3K27me3富集。令人意外的是，对rG4进行诱变并不会影响PRC2的招募，却会促进其在染色质上的释放与催化激活。但此时H3K27me3标记出现定位异常，基因沉默功能也受到损害。Xist-PRC2复合物会被困在S1染色体区室内，无法完成向S2染色体区室的必要转位。综上，Xist rG4的折叠动态调控了PRC2的活性、H3K27me3的富集，以及全染色体范围基因沉默的分步调控过程。