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HIRA-dependent H3.3 deposition and its modification H3.3K36me3 facilitate somatic hypermutation of immunoglobulin genes by maintaining the proper chromatin state and transcription

ABSTRACT: The H3.3 histone variant and its chaperone HIRA are involved in active transcription but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells is not yet fully understood. In this study we show that, knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of immunoglobulin heavy chain (IGH) in the human Ramos B cell line without changing the levels of AID and other major proteins known to be involved in SHM. Except for H3K79me2/3, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells and this was accompanied by decreased nascent transcription in IGH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the Ig locus was also reduced in the HIRA KO. Somewhat surprisingly HIRA loss increased the chromatin accessibility of Ig V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IGH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription coupled chromatin structure and accessibility contribute to SHM in human B cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180899 | GEO | 2021/07/29

REPOSITORIES: GEO

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Project description:Recognition of modified histones by “reader” proteins plays a critical role in the regulation of transcription1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions following RNA polymerase II (Pol II) elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin at an appropriate state to suppress cryptic transcription2,3. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies4. Here we show that the candidate tumor suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates Pol II elongation. Structural studies reveal that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific “Ser31” residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. ChIP-sequencing analysis reveal a genome-wide colocalization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription corepressor via modulating the transition of the promoter-proximal paused Pol II to elongation. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumor cell growth; higher expression of ZMYND11 is observed in triple-negative breast cancer patients with better prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth and tumor formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone variant-mediated transcription elongation control to tumor suppression. ChIP-seq analysis of ZMYND11, H3K36me3 in U2OS cells and ZMYND11 knockdown cells; ChIP-seq of H3.3 in Flag-H3.3 stable U2OS cells; RNA-seq of ZNYMD11 depleted U2OS cells.

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HIRA-dependent H3.3 deposition and its modification H3.3K36me3 facilitate somatic hypermutation of immunoglobulin genes by maintaining the proper chromatin state and transcription

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