BMI1 Is Recruited to DNA Breaks and Contributes to DNA Damage-Induced H2A Ubiquitination and Repair

Abstract

DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G 2 /M. These data support a crucial role for BMI1 in the cellular response to DNA damage.The induction of a DNA break leads to activation of multiple signaling pathways that lead to local modification of chromatin structure and recruitment of DNA repair complexes (18,22,55). Histone H2AX is rapidly phosphorylated near sites of DNA breaks by ATM, ATR, and DNA-PK (39, 54) and can spread to encompass a region of chromatin covering several megabases (40, 41).H2AX phosphorylation facilitates the recruitment of other proteins, including MDC1 (52) and the E3 ubiquitin ligases RNF8 and RNF168, which in turn participate locally in the K63-linked polyubiquitination of histones H2A and H2AX (23,32,50,51). Polyubiquitinated K63-linked histones provide a recognition element that recruits RAP80 through its ubiquitin interaction motifs (28,49,56). RAP80 can then promote the recruitment of other DNA repair factors such as BRCA1 and Abraxas, which are essential for efficient repair. RNF8 and RNF168 function are also required for proper localization of 53BP1, although the exact mechanism is unclear (12,23,32,51). 53BP1 recruitment to regions of DNA damage is dependent upon its Tudor domains, which have been found to specifically interact with methylated histone residues (6, 24, 42). A model has been proposed in which RNF8-and RNF168-mediated ubiquitination of histones confers local changes in chromatin structure, leading to exposure of methylated lysine residues in core histones, allowing the subsequent recruitment of 53BP1 (50). Enzymes involved in deubiquitination, such as BRCC36, USP3, and USP28, are also critical for efficient DNA repair, demonstrating that a dynamic regulation of ubiquitin conjugation and hydrolysis is necessary for optimal DNA repair (37,46,47,61).Polycomb group proteins BMI1 and RING1B/RNF2 form an active heterodimer E3 ligase that catalyzes the monoubiquitination of histone H2A at Lysine 119. (7,8,44,53,57). This activity is important for BMI1-mediated transcriptional silencing during organism development and cellular differentiation (27,48,58). Ubiquitination of H2A at lysine 119 is also induced locally at sites of DNA damage, both at s...

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

“…Focal accumulation of downstream factors RNF8 and 53BP1 were also reduced, as was DSB repair. At the extremes, mutation of histone H2AX lysines 119 and 120 to arginine and knockdown of RNF2 were both shown to inhibit histone H2AX phosphorylation (18,19), whereas at the other extreme, defects in signaling were not observed, but homologous recombination was impaired (15). The differences between these studies may reflect compensation by paralogues (10), differences in knockdown efficiency, and the potential reprogramming of gene expression that could accompany the lengthy incubation periods required in knockdown experiments.…”

A Novel Role of Chromodomain Protein CBX8 in DNA Damage Response

“…Interestingly, Bmi1 has also been reported to play a role in DNA damage repair and is also recruited to sites of laser scissors-induced local DNA breaks (19,52,53). However, Bmi1 has very different dynamics of recruitment to damage site from CBX8.…”

“…Recent studies have identified the role of polycomb group proteins such as Bmi1 in DNA damage response (19). Polycomb genes are thought to mediate transcriptional silencing and have been implicated in the control of embryogenesis, development, stem cell self-renewal, and heritable epigenetic states, as well as cell proliferation and cancer (1, 4, 20 -27).…”

Mitogen-activated Protein Kinase Signaling Mediates Phosphorylation of Polycomb Ortholog Cbx7

“…3A). PcG proteins and histones are also phosphorylated upon DNA damage (22,47). Therefore, we irradiated HEK 293 cells expressing Cbx7-FLAG to induce DNA doublestranded breaks.…”

“…Emerging evidence suggests that PcG proteins are regulated in response to the environment or cell signaling cues through post-translational modifications, particularly phosphorylation (21)(22)(23)(24). For example, phosphorylation of Mel-18 directs Ring1b to ubiquitylate H2A in chromatin (12), and Akt-mediated EZH2 phosphorylation at serine 21 results in decreased methyltransferase activity, allowing for derepression of silenced genes (25).…”