Abstract 2724: TGF-β signaling activity induces co-localization of both H4K20me3 and H3K27Ac at active enhancers during breast cancer epithelial-to-mesenchymal transition

Abstract In 2022, 2.3 million women worldwide were diagnosed with breast cancer, and more than670, 000 breast cancer-related deaths were reported. A leading contributor to breast cancer mortality is metastatic progression driven in part by the epithelial-to-mesenchymal transition (EMT). EMT is a reversible plastic state during which cancer cells spread beyond their primary environment. This plasticity in cell states can be mediated by epigenetic factors that influence gene expression through the regulation of chromatin accessibility. Our lab and others found that inhibition of Suv420H2/KMT5C, the methyltransferase that tri-methylates lysine 20 of histone H4 (H4K20), is typically associated with gene repression that promotes EMT in breast epithelial cells, suggesting that Suv420H2-mediating H4K20me3 plays a role in regulating epithelial vs. mesenchymal identity. To further dissect the role of H4K20me3 in EMT plasticity, MCF10Aimmortalized breast epithelial cells were treated with transforming growth factor-β1 (TGF-β1); a growth factor that acts as a tumor suppressor by limiting cell proliferation but can also promote tumor progression by driving EMT. Using a TGF-β treatment, recovery, and rechallenge approach followed by RT-qPCR analysis, I confirmed that TGF-β triggers EMTs by upregulating mesenchymal marker genes, such as EMT-transcription factors (ZEB1 and SNAI1/2), and downregulating epithelial marker genes (E-cad and EpCAM). In addition, cells gain a fibroblast-like morphology. TGF-β-induced EMT was reversible in response to TGF-β withdrawal during which cells regain their baseline morphology and re-express epithelial marker genes, showing a plastic state behavior. Additionally, we performed both a Cleavage Under Targets and Tagmentation and a Precision Run-On Sequencing on MCF10A cells to identify H4K20me3 distribution and nascent transcription activity. Collected data have revealed TGF-β-induced regions that gain H4K20me3, and these regions also acquire histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark associated with active promoter and enhancer regions in response to TGF-β. TGF-β-induced H4K20me3 and H3K27Ac co-localization sites are transcriptionally active and distal to transcriptional start sites, suggesting that they may be active enhancers. The significant accumulation and co-localization of both H4K20me3 and H3K27Ac at active enhancers in response to TGF-β suggests that H4K20me3 may have additional functions, beyond that of its known role as a repressor. A better understanding of the role of TGF-β-induced H4K20me3 sites at active enhancers in the context of EMT will provide insight to better target epigenetic modifiers to prevent breast cancer progression. Citation Format: Bachelard Dieujuste, Christina Davidson, Ching-Hua Shih, Laura McLane, Paula Vertino. TGF-β signaling activity induces co-localization of both H4K20me3 and H3K27Ac at active enhancers during breast cancer epithelial-to-mesenchymal transition [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 2724.