Promoter-proximal R-loops regulate binding of chromatin regulators and pluripotency [RIP-seq]

Numerous chromatin-remodelling factors are regulated by interactions with RNA. However, the contexts in which chromatin-remodelling factors encounter various RNA species, as well as the molecular functions of RNA binding, are poorly understood. Here we show that R-loops, RNA:DNA hybrids consisting of nascent transcripts hybridized to template DNA strands, facilitate embryonic stem cell (ESC) differentiation by modulating the binding of two key chromatin-remodelling enzymes near gene promoters. As previously shown for polycomb repressive complex 2 (PRC2)1-5, we find that the Tip60-p400 histone acetyltransferase and nucleosome-remodelling complex binds in cis to nascent transcripts. However, whereas chromatin binding by PRC2 is broadly inhibited by transcription6, transcription is necessary for maximal Tip60-p400 binding at most target loci. Given that nascent transcripts expressed from GC-rich promoters frequently form R-loops7, we mapped the genomic locations of R-loops in mouse ESCs, observing higher average Tip60-p400 levels and lower average PRC2 levels at genes with R-loops near their transcription start sites (TSSs). Disruption of R-loops by overexpression of RNaseH1 broadly reduced Tip60-p400 and increased PRC2 enrichment, demonstrating R-loops exert both positive and negative effects on chromatin association by regulatory factors. Consistent with these findings, RNaseH1 overexpression results in widespread changes in gene expression and inhibits ESC differentiation, allowing undifferentiated cells to persist for at least two weeks after differentiation is induced. These results define a novel mechanism by which promoter-proximal R-loops modulate chromatin structure to facilitate changes in cellular identity. We examined the interacting RNAs in Tip60-p400 complex by RIP-seq in mouse ES cells.

诸多染色质重塑因子均可通过与RNA的相互作用实现调控。然而，染色质重塑因子与各类RNA分子相遇的具体场景，以及RNA结合的分子功能，目前仍不甚明晰。本研究表明，R环（R-loop）——即新生转录本与模板DNA链杂交形成的RNA:DNA杂交双链——可通过调控两种关键染色质重塑酶在基因启动子附近的结合，促进胚胎干细胞（ESC）的分化。正如此前针对多梳抑制复合体2（PRC2）1-5的研究所示，我们发现Tip60-p400组蛋白乙酰转移酶与核小体重塑复合体可与新生转录本发生顺式结合。然而，与PRC2的染色质结合可被转录广泛抑制6不同，在大多数靶基因座上，转录是Tip60-p400实现最大程度结合的必要条件。鉴于GC富集启动子所表达的新生转录本常可形成R环7，我们对小鼠ESC中的R环基因组定位进行了绘制，发现在转录起始位点（TSS）附近存在R环的基因中，Tip60-p400的平均水平更高，而PRC2的平均水平更低。通过过表达核糖核酸酶H1（RNaseH1）破坏R环后，Tip60-p400的结合水平整体下降，而PRC2的富集程度则整体上升，这表明R环对调控因子的染色质结合兼具正向与负向调控作用。与上述发现一致，过表达RNaseH1会引发全基因组范围的基因表达改变，并抑制ESC分化，使得未分化细胞在诱导分化后至少可存活两周。上述结果揭示了一种全新的调控机制：启动子近端的R环可通过调控染色质结构，进而促进细胞身份的转变。我们通过RNA免疫共沉淀测序（RIP-seq），在小鼠ESC中检测了Tip60-p400复合体结合的RNA分子。