ChIP-seq for RNAPII before and after transcription inhibition

The FACT (FAcilitates Chromatin Transactions) complex is a conserved complex that maintains chromatin structure on transcriptionally active genes. Consistent with this, FACT is enriched on highly expressed genes, but how it is targeted to these regions is unknown. In vitro, FACT binds destabilized nucleosomes, supporting the hypothesis that FACT is targeted to transcribed chromatin through recognition of RNA polymerase-disrupted nucleosomes. In this study, we used high resolution analysis of FACT occupancy in S. cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and its interaction with chromatin is dependent on transcription by any of the three RNA polymerases. Deep sequencing of micrococcal nuclease (MNase)-resistant fragments shows that FACT-bound nucleosomes exhibit differing nuclease sensitivity compared to bulk chromatin, consistent with a modified nucleosome structure being the preferred ligand for this complex. Interestingly, a subset of FACT-bound nucleosomes may be “overlapping di-nucleosomes”, in which one histone octamer invades the ~147 bp territory normally occupied by the adjacent nucleosome. While the differing nuclease sensitivity of FACT-bound nucleosomes could also be explained by the demonstrated ability of FACT to alter nucleosome structure, transcription inhibition restores nuclease resistance suggesting that it is not due to FACT interaction alone. Collectively these results are consistent with a model in which FACT is targeted to transcribed genes through preferential interaction with RNA polymerase-disrupted nucleosomes.FACT（Facilitates Chromatin Transactions，染色质转录辅助复合体）是一类高度保守的复合体，可维持转录活跃基因区域的染色质结构。与此相符，FACT在高表达基因上呈现富集状态，但其被靶向至这些区域的具体机制仍未明确。体外实验显示，FACT能够结合不稳定的核小体，这为“FACT通过识别RNA聚合酶破坏的核小体，被靶向至转录中的染色质”这一假说提供了支持。本研究通过对酿酒酵母（S. cerevisiae）中FACT结合占据谱的高分辨率分析，对该假说进行了验证。研究证实，FACT在体内可与核小体发生相互作用，且其与染色质的结合依赖于三类RNA聚合酶任意一种介导的转录过程。对微球菌核酸酶（micrococcal nuclease, MNase）抗性片段的深度测序结果表明，与整体染色质中的核小体相比，FACT结合的核小体表现出差异化的核酸酶敏感性，这与“经过修饰的核小体结构是该复合体的优先结合配体”的观点一致。值得注意的是，一部分FACT结合的核小体可能属于“重叠双核小体”：即一个组蛋白八聚体侵入了原本由相邻核小体占据的约147bp的DNA区域。尽管FACT结合的核小体所表现出的异常核酸酶敏感性，也可通过FACT已被证实的改变核小体结构的能力加以解释，但转录抑制处理可恢复其核酸酶抗性，这表明该敏感性并非仅由FACT的相互作用单独导致。综合上述结果，本研究支持如下模型：FACT通过优先结合RNA聚合酶破坏的核小体，被靶向至转录活跃的基因区域。