Abstract
BackgroundThe histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks.ResultsOur comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. At transcription start sites, H3.3 dissociates rapidly with the highest rate at nucleosome-depleted regions (NDRs) just upstream of Pol II binding, followed by low H3.3 dissociation rates across gene bodies. H3.3 turnover at transcription start sites, gene bodies, and transcription end sites was positively correlated with transcriptional activity.H3.3 is found decorated with various histone modifications that regulate transcription and maintain chromatin integrity. We find greatly varying H3.3 dissociation rates across various histone modification domains: high dissociation rates at active histone marks and low dissociation rates at heterochromatic marks. Well- defined zones of high H3.3-nucleosome turnover were detected at binding sites of ESC-specific pluripotency factors and chromatin remodelers, suggesting an important role for H3.3 in facilitating protein binding. Among transcription factor binding sites we detected higher H3.3 turnover at distal cis-acting sites compared to proximal genic transcription factor binding sites.Our results imply that fast H3.3 dissociation is a hallmark of interactions between DNA and transcriptional regulators.ConclusionOur study demonstrates that H3.3 turnover and nucleosome stability vary greatly across the chromatin landscape of embryonic stem cells. The presence of high H3.3 turnover at RNA Pol II binding sites at extragenic regions as well as at transcription start and end sites of genes, suggests a specific role for H3.3 in transcriptional initiation and termination. On the other hand, the presence of well-defined zones of high H3.3 dissociation at transcription factor and chromatin remodeler binding sites point to a broader role in facilitating accessibility.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-8935-7-38) contains supplementary material, which is available to authorized users.
Highlights
The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification
ESCs that were routinely cultured in the absence of DOX were exposed to DOX-containing medium in order to inhibit HA-H3.3 expression and to measure H3.3 dissociation rates (Figure 1A)
HA-H3.3 ESC maintained their pluripotent state as demonstrated by the high expression of the pluripotency genes Oct4, Nanog and Sox2, which became downregulated upon embryoid body (EB) differentiation (Additional file 1: Figures S1 B and C)
Summary
The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks. Incorporation of histone variants into chromatin critically influences the properties of nucleosomes that play important roles in regulating transcription and epigenetic memory. The H3.3-specific chaperones AtrxDaxx and HIRA deposit H3.3 primarily at telomeres and non-heterochromatic regions, respectively [1,6,7]
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