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

ABSTRACT: 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. 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.Here, 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.Our 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.

SUBMITTER: Liao R

PROVIDER: S-EPMC5440973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Core histone variants, such as H2A.X and H3.3, serve specialized roles in chromatin processes that depend on the genomic distributions and amino acid sequence differences of the variant proteins. Modifications of these variants alter interactions with other chromatin components and thus the protein's functions. These inferences add to the growing arsenal of evidence against the older generic view of those linker histones as redundant repressors. Furthermore, certain modifications of specific H1 variants can confer distinct roles. On the one hand, it has been reported that the phosphorylation of H1 results in its release from chromatin and the subsequent transcription of HIV-1 genes. On the other hand, recent evidence indicates that phosphorylated H1 may in fact be associated with active promoters. This conflict suggests that different H1 isoforms and modified versions of these variants are not redundant when together but may play distinct functional roles. Here, we provide the first genome-wide evidence that when phosphorylated, the H1.4 variant remains associated with active promoters and may even play a role in transcription activation. Using novel, highly specific antibodies, we generated the first genome-wide view of the H1.4 isoform phosphorylated at serine 187 (pS187-H1.4) in estradiol-inducible MCF7 cells. We observe that pS187-H1.4 is enriched primarily at the transcription start sites (TSSs) of genes activated by estradiol treatment and depleted from those that are repressed. We also show that pS187-H1.4 associates with 'early estrogen response' genes and stably interacts with RNAPII. Based on the observations presented here, we propose that phosphorylation at S187 by CDK9 represents an early event required for gene activation. This event may also be involved in the release of promoter-proximal polymerases to begin elongation by interacting directly with the polymerase or other parts of the transcription machinery. Although we focused on estrogen-responsive genes, taking into account previous evidence of H1.4's enrichment of promoters of pluripotency genes, and its involvement with rDNA activation, we propose that H1.4 phosphorylation for gene activation may be a more global observation.

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

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