53BP1 regulates heterochromatin through liquid phase separation

Lei Zhang,Wenxiu Wang,Arishya Sharma,Fulai Jin,Sora Jin,Xiaoxia Guo,Youwei Zhang,Masaru Miyagi,Franklin Mayca Pozo,Yu Ichida,Mingyue Chen,Janet Wang,Yongjie Xu,Xinran Geng,Mingliang Tang,Jinshan Tang,Xinsheng Yao,Fangfang Wang,Michal Wozniak

doi:10.1038/s41467-022-28019-y

Abstract

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin protein HP1α in a mutually dependent manner. Deletion of 53BP1 results in a reduction in heterochromatin centers and the de-repression of heterochromatic tandem repetitive DNA. We identify domains and residues of 53BP1 required for its LLPS, which overlap with, but are distinct from, those involved in DSB repair. Further, 53BP1 mutants deficient in DSB repair, but proficient in LLPS, rescue heterochromatin de-repression and protect cells from stress-induced DNA damage and senescence. Our study suggests that in addition to DSB repair modulation, 53BP1 contributes to the maintenance of heterochromatin integrity and genome stability through LLPS.

Highlights

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; its involvement in other biological process is less well understood
When analyzing cellular localization of 53BP1, we found that 53BP1 formed nuclear puncta in MDA-MB-231 cells under normal growth conditions, which were not observed in 53BP1 KO cells (Fig. 1b), confirming that the nuclear puncta were 53BP1 signals in the absence of DNA damage
We found that the association of 53BP1 with SATIII was greatly reduced in heterochromatin protein 1α (HP1α)-depleted cells (Supplementary Fig. 10h), supporting the role of HP1α in promoting 53BP1’s association with heterochromatin

Summary

Introduction

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; its involvement in other biological process is less well understood. The formation of 53BP1 foci at DNA damage site is key for its function in DSB repair, which involves the OD-Tudor-UDR-NLS region[1,2]. ATM-dependent phosphorylation on multiple Ser/Thr residues at the N-terminus of 53BP1 is not required for its foci formation[10]; yet, it is crucial for recruiting 53BP1’s downstream factors including RIF1, PTIP, and the Shieldin complex to damage sites to promote the NHEJ pathway for DSB repair[11,12,13,14,15,16,17,18]. While facultative heterochromatin can be regulable by cell fate[19], constitutive heterochromatin is often permanent and forms at pericentromeric, centromeric and telomeric regions that include tandem repetitive DNA sequences[21] and/or transposable elements[22,23]. We report a DSB repair-independent role for 53BP1 in maintaining both the structural integrity and the transcriptional repression of heterochromatin through LLPS

Methods

Results

Conclusion