Mitotic H3K9 acetylation patterns are controlled by phase-specific activity of the histone deacetylases HDAC2, 3 and SIRT1

Histone acetylation play a fundamental role in many biological processes. H3K9ac is tightly linked to active transcription, and enriched in promoters, enhancers and insulators. During mitosis H3k9ac levels are reduced, and the mechanism of this reduction is not clear. Here, we used small molecules inhibitors of histone deacetylases and evaluated by immunofluorescence and western blots the involvement of each of the targeted enzymes in regulating H3K9ac during prophase, metaphase, anaphase, telophase and cytokinesis. We identified three histone deacetylases, HDAC2, HDAC3 and SIRT1, as modulators of H3K9ac mitotic levels. HDAC2 inhibition, increased global H3K9ac in prophase, whereas HDAC3 or SIRT1 inhibition, increased H3K9ac levels in metaphase. Next, we performed ChIP-seq in mitotic cells following specific inhibition of each of these histone deacetylases. While we observed a high concordance between the genomic areas impacted by HDAC2 and HDAC3, we detected a small set of loci that were unique to each condition, with HDAC3-specific targets being enriched for genes involved in mitosis regulation. Overall design: Examination of 4 different HDACs inhibitors in STC arrested mitotic Hela S3 cells. each experiment was done in duplicates and we also checked untreated mitotic cells and untreated unsynchronized cells

组蛋白乙酰化（histone acetylation）在诸多生物学过程中发挥核心调控作用。H3K9乙酰化修饰（H3K9ac）与活跃转录紧密关联，且富集于启动子、增强子及绝缘子区域。有丝分裂过程中，H3K9ac的水平会发生降低，其背后的具体调控机制目前尚未明确。
本研究采用组蛋白去乙酰化酶（histone deacetylase, HDAC）的小分子抑制剂，借助免疫荧光（immunofluorescence）与蛋白质印迹（western blots）技术，分析了各靶向酶在有丝分裂前期、中期、后期、末期及胞质分裂阶段对H3K9ac的调控作用。
本研究鉴定出HDAC2、HDAC3及SIRT1这3种组蛋白去乙酰化酶，为有丝分裂阶段H3K9ac水平的调控因子。
抑制HDAC2会使前期的整体H3K9ac水平升高，而抑制HDAC3或SIRT1则可提升中期的H3K9ac水平。
随后，我们分别对这3种组蛋白去乙酰化酶进行特异性抑制，并在有丝分裂细胞中开展了染色质免疫共沉淀测序（ChIP-seq）实验。
尽管HDAC2与HDAC3所调控的基因组区域具有高度一致性，但各处理组仍存在少量特有的基因座；其中HDAC3的特异性靶标显著富集于有丝分裂调控相关基因。
实验设计概述：本研究对经STC阻滞的有丝分裂HeLa S3细胞施加4种不同的HDAC抑制剂，所有实验均设置生物学重复；同时设置未处理的有丝分裂细胞与未同步化的未处理细胞作为对照。