Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

多能胚胎干细胞（pluripotent embryonic stem cells, ESCs）已知具有相对开放的染色质结构；尽管已有诸多研究试图解析ESCs的染色质特征，但染色质压缩在干细胞命运与功能中的作用仍不明确。连接组蛋白H1（linker histone H1）对高级染色质折叠至关重要，且对哺乳动物胚胎发生不可或缺。为探究H1与染色质压缩在干细胞多能性及分化中的作用，本研究对多种H1亚型缺失的胚胎干细胞的分化过程进行了分析。在去除白血病抑制因子（leukemia inhibitory factor）后，H1c/H1d/H1e三基因敲除ESCs在贴壁单层培养体系中的自发分化能力显著减弱。同样，多数三H1敲除胚胎体（embryoid bodies, EBs）无法形成代表三胚层的形态学结构，且仍保留未分化ESCs特有的基因表达特征。此外，在胚胎体进行神经分化时，三H1敲除细胞的神经突起生长存在缺陷，且无法有效激活神经标志物。最后，本研究发现，与野生型对照相比，三H1敲除胚胎及EBs无法完全抑制多能性基因的表达；且在干细胞分化与胚胎发生过程中，H1缺失会损害DNA甲基化，并改变启动子区域的组蛋白修饰，而这些修饰是有效沉默多能性基因Oct4所必需的。综上，本研究证实H1在多能干细胞分化中发挥关键作用，研究结果提示H1与染色质压缩可能通过表观遗传调控多能性基因的沉默，介导多能干细胞的分化过程。