Abstract
BackgroundStructural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells.ResultsHere, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18.ConclusionsOur data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance.
Highlights
Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes
Site‐specific changes in global H1 phosphorylation during cell differentiation We have generated a collection of highly specific antisera, raised against synthetic phosphopeptides, which recognize phosphorylation at single sites that are unique to individual human H1 variants or are shared between just two variants
In this study, we demonstrated that interphase phosphorylation of H1 variants is reduced preferentially at specific sites during pluripotent cell differentiation and that the enrichment of pS187-H1.4, but not pS18-H1.5, is positively correlated with transcription
Summary
Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Human cells differentially express 11 genes encoding non-allelic amino acid sequence variants of H1 [4, 5]. These share a common tripartite structure in which a conserved globular domain (GD) is flanked by a short N-terminal domain (NTD) and a longer C-terminal domain (CTD) [6]. Analyses of synchronized cells from multiple organisms suggest that H1 phosphorylation increases progressively during interphase before peaking transiently during mitosis [14,15,16,17,18,19] These early studies did not identify which sites in individual H1 variants are phosphorylated during interphase and mitosis. S18, S173 and S189 were identified as sites of interphase phosphorylation of H1.5 [21, 24], but the significance of these phosphorylations remains unclear [13]
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