Critical Role of Monoubiquitination of Histone H2AX Protein in Histone H2AX Phosphorylation and DNA Damage Response

Abstract

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (␥-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of ␥-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers. DNA damage response (DDR)3 involves serial molecular events that are activated during DNA double strand breaks (DSBs) and are important for the repair of damaged DNA (1, 2). Histone variant H2AX is a central player for DDR. Upon DNA damage, H2AX is phosphorylated by ataxia telangiectasia mutated (ATM) and ATM-related kinases at serine 139, known as ␥-H2AX, which serves as a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) to sites of DNA damage, named DNA damage foci (3, 4). MDC1 is then phosphorylated by ATM within the DNA damage foci and facilitates the recruitment of RING finger protein 8 (RNF8) ubiquitin ligase (E3) to the DNA damage foci (1, 2). RNF8 then recruits RING finger protein 168 (RNF168) E3 ligase to promote polyubiquitination of proteins near the DNA damage foci, in turn recruiting breast cancer 1, early onset (BRCA1)-receptor-associated protein 80 (RAP80) complex to DNA damage sites for DNA repair.

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.…”

“…There have been considerable differences in the reported consequences of BMI1 knockdown on the recruitment of DDR proteins to IRIFs (14,16,18,19,24,25). The recruitment of phosphorylated ATM to IRIFs, for example, differed between laboratories.…”

The emerging role of Polycomb repressors in the response to DNA damage

“…This raises the possibility that the PRC1 E3 ligase is also involved in DDR. Several laboratories confirmed this hypothesis by showing that both BMI1 and RING1B efficiently accumulate at ionizing radiation (IR)-and laser-induced DSBs (Chagraoui et al, 2011;Chou et al, 2010;Facchino et al, 2010;Gieni et al, 2011;Ginjala et al, 2011;Ismail et al, 2010;Nacerddine et al, 2012;Pan et al, 2011;Wu et al, 2011a) (Box 1 and Fig. 3).…”

“…The role of RING1B and BMI1 in DSB-induced histone ubiquitylation has been addressed specifically, both biochemically and by using immunofluorescence. Immunoblot analysis of H2A and H2AX suggests that loss of RING1B or BMI1, or mutation of the canonical target lysine of H2A or H2AX interfere with both basal and IR-induced monoubiquitylation (Ginjala et al, 2011;Ikura et al, 2007;Pan et al, 2011;Wu et al, 2011a). In addition, ubiquitylation of H2A -as detected using the antibody E6C5 -at laser-induced damage is found to depend on BMI1 (Ginjala et al, 2011).…”

Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

“…A3A expression also caused increased levels of the DNA damage marker ␥-H2AX. Interestingly, the increase was most obvious for the monoubiquitinated form of ␥-H2AX, which is an important mediator of the DNA damage response (41)(42)(43). In contrast, endogenous A3A was not toxic, despite the high levels of A3A induction by type I IFN seen in primary CD14…”