Control of Regulatory Element Function by Histone H3.3




Tafessu, Amanuel Melesse

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In eukaryotic cells, DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. While chromatin functions in part to organize a large amount of genomic material within the confines of the nucleus, regulatory DNA sequences consequently become masked to transcription machinery. Such regulatory sequences are enriched in specific histone variants and post-translational modifications (PTMs). The histone variant H3.3 is enriched at transcriptionally active regulatory elements such as promoters and enhancers. While recent studies have revealed a role for H3.3 in silencing repetitive elements and repressing developmentally regulated promoters, it is unclear how H3.3 contributes to chromatin states at active promoters and enhancers. In this study, we performed genomic analyses of chromatin features associated with active regulatory elements in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread dysregulation in the absence of H3.3. Loss of H3.3 or HIRA- the chaperone responsible for H3.3 deposition to transcriptionally active regions- reduces chromatin accessibility and transcription factor (TF) footprinting at promoters. Further, H3.3 KO ESCs show reduced promoter enrichment of p300- a transcriptional coactivator responsible for H3 acetylation at lysine 27 (H3K27ac). Consequently, H3.3 KO ESCs show reduced H3K27ac at promoters, along with reduced enrichment of the acetyllysine reader BRD4. Despite the enrichment of H3.3 at both promoters and enhancers, it appears to play distinct roles at these regions. ESCs lacking H3.3 or HIRA are able to maintain both accessibility and TF footprinting at enhancers, but still show reduced H3K27ac. Unlike promoters, enhancers show no deficit of p300 enrichment in the absence of H3.3. The loss of H3K27ac observed at enhancers of H3.3 KO ESCs can be attributed to reduced catalytic activity of p300. In particular, phosphorylation of Ser31, the only residue unique to the N-terminal tail of H3.3, facilitates p300 activity and H3K27ac enrichment. In spite of extensive chromatin dysregulation and reduced active RNA polymerase II (RNAPII) engagement, ESCs maintain transcription from ESC-specific genes in the absence of H3.3. However, H3.3 KO ESCs are unable to initiate lineage-specific transcription upon undirected differentiation. In line with their differentiation defect, H3.3 KO ESCs retain footprinting of ESC-specific TFs and fail to generate footprints of lineage-specific TFs. Further, H3.3 KO ESCs fail to "open" and acetylate developmentally regulated enhancers. Overall, our study shows that H3.3 facilitates the establishment of transcriptionally permissive chromatin at regulatory elements, with context-dependent outcomes for transcriptional output. While H3.3 is not required for maintaining transcription in ESCs, it plays a key role in activating promoters and enhancers during differentiation.

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