Abstract 2750: Alternative enhancer usage of <i>PER2</i> locus regulating 3D genome architecture in epithelial-mesenchymal states

Abstract The regulation of epithelial (E) and mesenchymal (M) states requires orchestrated adaptation at the epigenomic level. The 3D genome hierarchical orders, together with chromatin accessibility, histone modifications, and local DNA looping, establish the architectural backbone of the genome in response to functional ques for EM plasticity during cancer progression. Here, we delved into alternative enhancer usage and the accompanied changes in 3D genome architecture which could govern the interconversion between these two states. Using ovarian cancer (OC) cells as a model, distinct enhancer usage at the circadian gene Period 2 (PER2) loci was identified in OC cells of epithelial-like Epi-A (PEO1 and OVCA429) and mesenchymal-like Mes (SKOVE3 and HeyA8) subtypes. Intron 6-14 of PER2 consisted of multiple enhancers in PEO1 and OVCA429 suggesting an open chromatin state compared to a relatively closed chromatin in SKOV3 and HeyA8 marked by latent/inactive regions. OC tumors of epithelial-like Epi-A subtype with high PER2 expression were also correlated with better overall survival, suggesting the clinical relevance of this region. CRISPR/Cas9-mediated deletion of 3 enhancers, namely A, B, and C regions, was performed to generate clones in OVCA429. Deletion of specific enhancer B and C regions of PER2 resulted in a spindly mesenchymal morphology, compared to the cobblestone epithelial morphology of parental OVCA429 and clones with region A deletion. These changes corresponded with the altered expression of known EMT markers. RNA-seq analysis showed a significant shift in the EMT scores of OVCA429_PER2_B towards a Mes-like profile. Hi-C analysis was done in OVCA429_PER2_B to decipher the 3D genome architecture. Changes at compartments, topologically associated domains (TADs), and long-range DNA interaction loops were explored. Our findings highlighted that alternative enhancer usage is crucial in EM plasticity and subtype conversion, offering genome level insights into EMT regulatory mechanisms in cancers. Citation Format: Yi-Chia Chiu, Qing You Pang, Tuan Zea Tan, Wendy W. Hwang-Verslues, Ruby Y. J. Huang. Alternative enhancer usage of PER2 locus regulating 3D genome architecture in epithelial-mesenchymal states [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 2750.