Histone Deacetylases (HDACs) are essential for transcription of the pluripotent network in embryonic stem cells [Pro-Seq]

Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening and therefore HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of HDAC1 and HDAC2 in embryonic stem cells (ESC) reduced expression of pluripotent transcription factors, Oct4, Sox2 and Nanog (OSN). By shaping global histone acetylation HDACs indirectly regulate the activity of acetyl-lysine readers, such as BRD4. To examine the direct effects of HDAC (LBH589) and BRD4 (JQ1) inhibition on the ESC transcriptome, we performed precision nuclear run-on and sequencing (PRO-seq) analysis. Both LBH589 and JQ1 caused a marked reduction in the pluripotent network. However, while JQ1 treatment induced widespread transcriptional pausing of genes, HDAC inhibition caused a reduction in both paused and elongating polymerase, suggesting an overall reduction in recruitment of RNAPII. Using enhancer RNA (eRNA) expression to measure enhancer activity, we found that while HDAC activity regulates fewer enhancers than JQ1, LBH589 sensitive eRNAs were preferentially associated with super-enhancers and OSN binding sites. These findings suggest that HDAC activity is required for maintenance of pluripotency by regulating the OSN enhancer network via optimal recruitment of RNA polymerase II. Overall design: To examine the direct effects of HDAC (LBH589) and BRD4 (JQ1) inhibition on the ESC transcriptome, we performed precision nuclear run-on and sequencing (PRO-seq) analysis.

组蛋白乙酰化是一种动态的表观遗传修饰，其过程由组蛋白乙酰转移酶与组蛋白去乙酰化酶（HDACs）的拮抗作用共同调控。组蛋白尾端的去乙酰化会导致染色质浓缩，因此HDACs通常被认为是转录抑制因子。但与直觉相悖的是，在胚胎干细胞（ESC）中同时敲除HDAC1与HDAC2，会导致多能转录因子Oct4、Sox2及Nanog（OSN）的表达水平下降。
HDACs通过调控全局组蛋白乙酰化分布，间接调节乙酰赖氨酸阅读器（如BRD4）的活性。
为探究HDAC抑制剂LBH589与BRD4抑制剂JQ1对ESC转录组的直接调控作用，我们开展了精确核连缀测序（PRO-seq）分析。LBH589与JQ1处理均显著削弱了多能性调控网络。然而，JQ1处理会引发基因广泛的转录暂停，而HDAC抑制则同时降低了暂停状态与延伸状态的RNA聚合酶II（RNAPII）水平，提示RNAPII的招募整体减少。
我们以增强子RNA（eRNA）的表达水平衡量增强子活性，结果发现：尽管HDAC调控的增强子数量少于JQ1，但对LBH589敏感的eRNA优先与超级增强子及OSN结合位点相关联。
上述结果表明，HDAC活性可通过最优招募RNA聚合酶II，调控OSN相关增强子网络，从而维持胚胎干细胞的多能性。
整体实验设计：为探究HDAC抑制剂LBH589与BRD4抑制剂JQ1对ESC转录组的直接调控作用，我们开展了精确核连缀测序（PRO-seq）分析。