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PRC2-indepdendent actions of H3.3K27M in embryonic stem cell differentiation

ABSTRACT: The histone H3 variant, H3.3, is localized at specific regions in the genome, especially promoters and active enhancers, and has been shown to play important roles in development. A lysine to methionine substitution in position 27 (H3.3K27M) is a main cause of Diffuse Intrinsic Pontine Glioma, a lethal type of pediatric cancer. H3.3K27M has a dominant-negative effect by inhibiting the Polycomb Repressor Complex 2 (PRC2) activity. Here, we studied the immediate, genome-wide, consequences of the H3.3K27M mutation independent of PRC2 activity. We developed Doxycycline (Dox)-inducible mouse embryonic stem cells (ESCs) carrying a single copy of WT-H3.3, H3.3K27M and H3.3K27L, all fused to HA. We performed RNA-Seq and ChIP-Seq at different timepoints following Dox in undifferentiated and differentiated ESCs. We find increased binding of H3.3 around transcription start sites in cells expressing both H3.3K27M and H3.3K27L compared with WT, but not in cells treated with PRC2 inhibitors. Differentiated cells carrying either H3.3K27M or H3.3K27L retain expression of ESC-active genes, in expense of expression of genes related to neuronal differentiation. Taken together, our data suggest that a modifiable H3.3K27 is required for proper histone incorporation and cellular maturation, independent of PRC2 activity.

ORGANISM(S): Mus musculus

PROVIDER: GSE195789 | GEO | 2022/09/01

REPOSITORIES: GEO

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Project description:Histone variants and their chaperones are key regulators of eukaryotic transcription, and are critical for normal development. The histone variant H3.3 has been shown to play important roles in pluripotency and differentiation, and although its genome-wide patterns have been investigated, little is known about the role of its dynamic turnover in transcriptional regulation. To elucidate the role of H3.3 dynamics in embryonic stem cell (ESC) biology, we generated mouse ESC lines carrying a single copy of a doxycycline (Dox)-inducible HA-tagged version of H3.3, and monitored the rate of incorporation of the HA-tagged variant by ChIP-seq at varying time points following Dox induction. Active H3K4me3-enriched genes display high turnover dynamics in transcription start sites (TSS); H3K27me3-enriched genes have a lower turnover dynamics in the TSS, but higher turnover rates in gene bodies; developmental bivalent genes marked by both H3K4me3 and H3K27me3 show a mixed behavior with high TSS dynamics and high gene body dynamics, especially in genes bound by both PRC1 and PRC2. In contrast, genes proximal to super-enhancers show the most reduced gene-body turnover dynamics, while genes proximal to other enhancers show no unique pattern. In RA-treated cells, while the overall profile in expressed genes remains largely unaltered over the TSS and gene body, a significant and dramatic decrease in turnover occurs at the -1 nucleosome position immediately before the TSS, revealing a hyperdynamic nucleosome in promoters of genes expressed in ESCs. We suggest that histone turnover dynamics provides an additional mechanism involved in expression regulation, that gene-body turnover dynamics is distinct from that of promoter regions, and that a hyperdynamic -1 nucleosome marks promoters in ESCs. Our data provide evidence for regional regulation of H3.3 turnover in ESC promoters, and calls for testing, in high resolution, the dynamic behavior of additional histone variants and other structural chromatin proteins.

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PRC2-indepdendent actions of H3.3K27M in embryonic stem cell differentiation

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