The histone deacetylase SIRT6 controls transcription elongation via promoter- proximal pausing (ChIP-Seq)

Transcriptional regulation in eukaryotes commonly occurs at promoter-proximal regions wherein transcriptionally engaged RNA Polymerase II (Pol II) pauses before proceeding towards productive elongation. The roles of chromatin in this process remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 regulates transcription elongation by binding to Pol II and anchoring the Negative ELongation Factor NELF, thereby preventing the release of Pol II towards elongation. Absence of SIRT6, or conditions of glucose deprivation, lead to increased levels of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of the CDK9 kinase, phosphorylation of NELF and Pol II, and recruitment of the transcription factors MYC and BRD4, the PAF1 Complex, as well as several positive elongation factors, in turn releasing Pol II into productive elongation. Collectively, we identified SIRT6 as the first pausing-dedicated histone deacetylase, regulating intragenic H3K9ac and H3K56ac as critical chromatin modifications modulating transcriptional pausing and elongation. ChIP-seq experiments to determine genome wide profiles of multiple histone modifications (H3K36me3, H3K79me2, H3K9ac,H3K56ac), RNA polymerase II and the transcription factor LEO1 component of the PAF1 complex) in WT and SIRT6 KO mouse embryonic stem cells (129 mouse strain).

真核生物的转录调控通常发生在启动子近端区域，此时处于转录结合状态的RNA聚合酶II（RNA Polymerase II）会在进入有效延伸阶段前发生暂停。目前学界对染色质在该过程中的作用仍知之甚少。本研究证实，组蛋白去乙酰化酶SIRT6可通过结合RNA聚合酶II并锚定负延伸因子NELF（Negative ELongation Factor），调控转录延伸过程，进而阻止RNA聚合酶II释放进入延伸阶段。当SIRT6缺失或细胞处于葡萄糖剥夺条件时，组蛋白H3第9位赖氨酸乙酰化（H3K9ac）与第56位赖氨酸乙酰化（H3K56ac）的水平会升高，CDK9激酶被激活，NELF与RNA聚合酶II发生磷酸化，同时招募转录因子MYC、BRD4、PAF1复合物（PAF1 Complex）以及多种正向延伸因子，最终促使RNA聚合酶II释放并进入有效延伸阶段。综上，本研究鉴定出SIRT6是首个专司转录暂停调控的组蛋白去乙酰化酶，其通过调控基因内部区域的H3K9ac与H3K56ac这两种关键染色质修饰，进而调节转录暂停与延伸过程。本研究开展了染色质免疫共沉淀测序（ChIP-seq）实验，以解析野生型（wild type, WT）与SIRT6敲除（knockout, KO）129品系小鼠胚胎干细胞中，多种组蛋白修饰（H3K36me3、H3K79me2、H3K9ac、H3K56ac）、RNA聚合酶II以及PAF1复合物组分LEO1的全基因组分布特征。