Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0135-3) contains supplementary material,...

Site-specific phosphorylation of histone H1.4 is associated with transcription activation

“…These data are consistent with a previous report by our lab, in which pS187-H1.4 was identified as a bonafide substrate for the kinase activity of CDK9 [9], and further verifies the pS187-H1.4 signal. Table 2.…”

“…In a more recent study, we showed that the global levels of H1 phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and pS187-H1.4 are subject to differential regulation and that CDK9 phosphorylated pS187-H1. 4 was associated with maintenance of pluripotency [9]. This provided the first evidence that pS187-H1.4 is may be important for transcriptional activation.…”

“…H1.4 undergoes various post-translational modifications (PTMs), such as K26 methylation [41] which allows binding of HP1 and subsequent heterochromatin function, and Gcn5-mediated H1.4K34Ac [42], which works to activate transcription by facilitating the binding of chromatin remodelers and recruitment of transcription factors. Furthermore, ChIP and qRT-PCR studies from our laboratory in Hela, NT2 and mESC cells have implicated pS187-H1.4 in transcriptional activation, showing enrichment of this modified variant at promoters of active genes [8,9].…”

“…We previously generated a collection of unique, highly specific antisera using synthetic phosphopeptides and recombinant proteins and demonstrated their ability to recognize phosphorylation at single sites [8,9]. These sites are either exclusive to an individual human H1 variant or are shared between only two variants.…”

“…Chromatin immunoprecipitation was performed using protocols described before [9] with minor adjustments. Briefly, cells were cross-linked using final concentration 1% methanol-free paraformaldehyde (Thermo Fisher Scientific) for 8 minutes quenched by 125mM final concentration glycine for 10 minutes.…”