Abstract
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.
Highlights
Pluripotent embryonic stem cells (ESCs) can self-renew and differentiate into diverse cell types, including lineages from all three germ layers present in the adult organism, offering great promise in regenerative medicine in addition to serving as a useful system for developmental biology studies
By analyzing the differentiation capacity of embryonic stem cells (ESCs) that lack multiple H1 subtypes, we find, for the first time, that H1 and higherorder chromatin compaction are required for proper differentiation and lineage commitment of pluripotent stem cells
We present evidence that H1 participates in mediating changes of histone marks and DNA methylation necessary for silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis
Summary
Pluripotent embryonic stem cells (ESCs) can self-renew and differentiate into diverse cell types, including lineages from all three germ layers present in the adult organism, offering great promise in regenerative medicine in addition to serving as a useful system for developmental biology studies. Recent reports have associated ESCs with a open, hyperdynamic chromatin and hyperactive global transcription [2,5,6], and open chromatin has been suggested as a marker for pluripotency [7,8]. It remains undetermined whether higher order chromatin compaction is required for pluripotent stem cell differentiation and how an open chromatin state impacts stem cell function. The basic repeating unit of chromatin is the nucleosome core particle, which consists of an octamer of four core histones (H2A, H2B, H3 and H4)
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