Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA, 30602, USA.
Regenerative Biosciences Center (RBC), University of Georgia, Athens, GA, 30602, USA.
Epigenetics Chromatin. 2021 Dec 27;14(1):58. doi: 10.1186/s13072-021-00432-5.
Directed differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.
直接诱导分化小鼠胚胎干细胞（mESCs）或诱导多能干细胞（iPSCs）为解析导致特定细胞谱系形成的分子机制提供了强大的模型。组蛋白去乙酰化酶抑制剂的处理可以显著提高定向分化的效率。然而，其机制尚不清楚。在这里，我们使用 CUT&RUN 联合 ATAC-seq 来确定 VPA 暴露后组蛋白修饰和全基因组染色质可及性的变化。VPA 诱导了全局组蛋白 H3 乙酰化（H3K56ac）的显著增加，这是一种影响核小体稳定性的核心组蛋白修饰，以及与细胞骨架组织和细胞形态发生相关的基因座的富集。此外，VPA 改变了连接组蛋白 H1 亚型的水平和总组蛋白 H1/核小体的比值，表明初始分化事件。值得注意的是，ATAC-seq 分析显示，参与调节 CDK 丝氨酸/苏氨酸激酶活性和 DNA 双链解旋的基因的染色质可及性发生了变化。重要的是，在几个关键的基因组基因座，如多能因子 Left、心肌肌钙蛋白 Tnnt2 和同源盒因子 Hopx，可见染色质可及性的变化，这些基因座在心肌细胞分化中起着关键作用。大规模平行转录因子（TF）足迹分析也表明，参与向中胚层和内胚层谱系分化的 TF 的占有率增加，以及 VPA 处理后 POU5F1/ SOX2 多能因子足迹的丧失。我们的结果提供了 VPA 诱导 mESCs 分化后染色质景观的首次全基因组分析，并为调控多能性丧失和早期谱系决定的复杂分子过程提供了新的机制见解。
