Abstract 1720: 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS)

Abstract As compacted DNA, heterochromatin represses abnormal gene expression by inhibiting DNA transcription and maintains genome integrity by protecting aberrant chromosome segregation. Therefore, abnormalities in heterochromatin function are commonly found in cancer. However, how exactly heterochromatin preserves the genome integrity remains poorly understood due largely to the complex structure of heterochromatin. Recently, increasing evidence suggests that heterochromatin factors, including HP1α, KAP1, and SUV39H1/2, could drive the soluble heterochromatin into phase-separated droplets/condensates to promote gene silencing, which is referred to as liquid-liquid phase separation (LLPS). TP53-binding protein 1 (53BP1) has traditionally been a critical factor in DNA double-strand break (DSB) repair. We recently reported an unexpected finding that under normal growth conditions, 53BP1 regulates heterochromatin function through LLPS, which is independent of its DSB repair function. Our studies reveal a new paradigm in studying 53BP1, heterochromatin, and genome stability. However, an important question remains unanswered: how 53BP1 is recruited to heterochromatin to undergo LLPS and the associated broad biological impact. To address this question, we performed a special crosslinking coupled mass spectrometry to uncover potential factors in the 53BP1 condensates assembled on heterochromatin. We identified many factors associated with DNA methylation and histone modification, which are the two main steps in restoring heterochromatin epigenetic modification. Their interactions with 53BP1 at heterochromatin were confirmed by different approaches. We further focused on one of the key factors, PCNA, and carried out a detailed analysis of the interaction between PCNA and 53BP1 and the role of such interaction in 53BP1 LLPS, DSB repair, and heterochromatin function. These findings reveal the previously uncharacterized function of 53BP1 at heterochromatin during normal DNA replication. Citation Format: Xinran Geng, Youwei Zhang. 53BP1 regulates heterochromatin through liquid-liquid phase separation (LLPS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1720.