Abstract
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.
Highlights
Histone H1 and the linker histones are members of a family of lysine-rich proteins that have classically been identified as structural components, playing a role in the formation of higher order chromatin structures associated with inaccessibility to transcriptional machinery [1,2]
In a more recent study, we showed that the global levels of H1 phosphorylation at H1.5 H1.5-Ser18, H1.2/H1.5-Ser173 and pS187-H1.4 are subjected to differential regulation, and that pS187-H1.4 was associated with the maintenance of pluripotency [19]
While the phospho-H1 antisera provide a measure of phosphorylation between specific sites, the pan-H1 antisera provide a comparison of the H1 variant amounts present, regardless of their phosphorylation status
Summary
Histone H1 and the linker histones are members of a family of lysine-rich proteins that have classically been identified as structural components, playing a role in the formation of higher order chromatin structures associated with inaccessibility to transcriptional machinery [1,2]. Based on older chromatography reports and more recent mass spectrometry data, phosphorylation is observed to be the most abundant modification, progressively increasing during the cell cycle and transiently peaking at mitosis [11,12,13] These studies revealed that different residues are phosphorylated during interphase and mitosis and identified the specific sites of phosphorylation within the CTD. We showed that pS187-H1.4 stably binds RNAPII and co-localizes with active RNAP II peaks, suggesting a functional interaction between the two proteins These signals were corroborated by a GRO-seq expression dataset to show that the active pS187-H1.4 signals were seen at the promoters of genes undergoing transcriptional elongation. These data provide evidence for a more nuanced role of H1 and put forth the possibility of a new layer of regulation within the accepted model of transcriptional activation
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