Abstract 2743: The H2B-E76K oncohistone alters chromatin accessibility, composition, and gene expression to induce EMT

Abstract Background: Histones are essential nucleosome components that regulate DNA accessibility and transcription. Mutations in histones, such as H2B-E76K, are prevalent in various cancers, including lung and genitourinary cancers. The H2B-E76K mutation destabilizes nucleosomes by disrupting H2A/H2B dimer binding to H3/H4 tetramers, altering chromatin structure and function. Objectives: Investigate the effects of the H2B-E76K mutation on gene expression, chromatin interactions, signaling pathways, and cellular phenotypes, focusing on the induction of epithelial-mesenchymal transition (EMT) in lung cancer models. Methods: CRISPR-Cas9 created BEAS-2B bronchial epithelial cell lines with heterozygous and homozygous H2B-E76K mutations in the H2BC4 gene. Phenotypic assays assessed cell growth, migration, invasion, and EMT marker expression. Transcriptomic changes were analyzed using scRNA-seq, bulk RNA-seq, and PRO-seq to evaluate pathway deregulation, transcriptional instability, and RNA polymerase activity. The μMap technique mapped protein-chromatin interactions disrupted by the mutation. Chromatin profiling with CUT&RUN and ATAC-seq examined histone incorporation and chromatin accessibility changes. Results: H2B-E76K mutant cells showed enhanced migration and invasion, increased mesenchymal markers (N-cadherin, vimentin), and decreased epithelial markers (E-cadherin), consistent with EMT. These cells also resisted apoptosis upon growth factor withdrawal. Transcriptomic analyses revealed transcriptional instability, with increased cell-to-cell variability in gene expression, altered patterns linked to oncogenic signaling, transcript switching, and disrupted RNA polymerase activity. The μMap technique identified disrupted interactions between chromatin and RNA-processing enzyme RAVER1, correlating with altered splicing, exon skipping, and gene expression variability. ATAC-seq showed enhanced binding of EMT-related transcription factors (e.g., AP1, CEBPB, ZNF384, FOXC2) and increased chromatin accessibility at EMT-related promoters. CUT&RUN revealed diffuse genome-wide deposition of H2B-E76K histones, consistent with replication-coupled incorporation. These chromatin changes promoted transcriptional programs associated with EMT and cancer progression. Conclusions: H2B-E76K histones are broadly distributed throughout the genome, causing transcriptomic instability, altered splicing, and transcription factor binding changes. These alterations drive EMT phenotypes, including enhanced invasion, migration, and resistance to apoptosis, contributing to aggressive cancer behavior. H2B-E76K acts as a key epigenetic driver of oncogenesis by modifying chromatin accessibility, gene regulation, and cellular plasticity. Citation Format: Pierre Priam, Kimberly Espinoza Pereira, Jixiu Shan, Anthony Lamberto, Jay Sarthy, Wuyue Zhou, Ciaran Seath, Richard L. Bennett, Jonathan D. Licht. The H2B-E76K oncohistone alters chromatin accessibility, composition, and gene expression to induce EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2743.