Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168

Abstract

The mammalian E3 ubiquitin ligases RNF8 and RNF168 facilitate recruitment of the DNA damage response protein 53BP1 to sites of DNA double-strand breaks (DSBs). The mechanism involves recruitment of RNF8, followed by recruitment of RNF168, which ubiquitinates histones H2A/H2AX on K15. 53BP1 then binds to nucleosomes at sites of DNA DSBs by recognizing, in addition to methyl marks, histone H2A/H2AX ubiquitinated on K15. We report here that expressing H2AX fusion proteins with N-terminal bulky moieties can rescue 53BP1 recruitment to sites of DNA DSBs in cells lacking RNF8 or RNF168 or in cells treated with proteasome inhibitors, in which histone ubiquitination at sites of DNA DSBs is compromised. The rescue required S139 at the C-terminus of the H2AX fusion protein and was occasionally accompanied by partial rescue of ubiquitination at sites of DNA DSBs. We conclude that recruitment of 53BP1 to sites of DNA DSBs is possible in the absence of RNF8 or RNF168, but still dependent on chromatin ubiquitination.

Chronic oxidative stress promotes H2 AX protein degradation and enhances chemosensitivity in breast cancer patients

“…Thus, under chronic oxidative stress, RNF168 might interact with K13–K15 residues of H2AX protein and further promote binding of a yet‐unidentified ubiquitin ligase on K119, facilitating H2AX degradation. This model is in complete agreement with previously published results in the field (Pinato et al , ; Mattiroli et al , ; Kocylowski et al , ) and suggests for the first time a connection between RNF168 and K119 ubiquitination under chronic oxidative stress, which mechanism will be interesting to delineate in future work. Although RNF168‐mediated K13–K15 ubiquitination is well established to facilitate 53BP1 binding to DSB sites, the role of K119 ubiquitination is less clear.…”

“…In addition to its role in BC tumorigenesis and chemosensitivity, our work brings new insights into the role of RNF168, as it indicates that RNF168 not only participates in DDR signalling, as shown previously (Doil et al , ; Pinato et al , ; Stewart et al , ; Ismail et al , ; Ginjala et al , ; Gatti et al , ; Mattiroli et al , ; Bohgaki et al , ; Chen et al , ; Fradet‐Turcotte et al , ; Leung et al , ; Kocylowski et al , ), but is also involved in H2AX stability under chronic stress. Indeed, the concerted action of the E3 ubiquitin ligases RNF8 and RNF168 upon DNA damage is well known to contribute to the activation of double strand break (DSB) repair pathway (Pinato et al , ; Mattiroli et al , ; Kocylowski et al , ). RNF8 activation at the site of DSB is required for the recruitment of RNF168, which specifically mono‐ubiquitinates K13 and K15 residues of H2A‐type histones and induces conjugation of K63‐linked ubiquitin chains on these lysines to activate DDR signalling (Pinato et al , ; Mattiroli et al , ; Kocylowski et al , ).…”

“…We then looked for E3 ubiquitin ligases that might be implicated in this process. In particular, the E3 ubiquitin ligases RNF8, RNF168 and RING1B/BMI1 that are known to ubiquitinate H2AX on K13/15 and K119, respectively, although these enzymes have until now been strictly involved in the activation of the DDR (Kolas et al , ) (Doil et al , ; Pinato et al , ; Stewart et al , ; Ismail et al , ; Ginjala et al , ; Gatti et al , ; Mattiroli et al , ; Bohgaki et al , ; Chen et al , ; Fradet‐Turcotte et al , ; Jackson & Durocher, ; Leung et al , ; Kocylowski et al , ). We thus evaluated whether the silencing of any of these enzymes might affect H2AX‐WT protein stability.…”

“…We then looked for E3 ubiquitin ligases that might be implicated in this process. In particular, the E3 ubiquitin ligases RNF8, RNF168 and RING1B/ BMI1 that are known to ubiquitinate H2AX on K13/15 and K119, respectively, although these enzymes have until now been strictly involved in the activation of the DDR ( Leung et al, 2014;Kocylowski et al, 2015). We thus evaluated whether the silencing of any of these enzymes might affect H2AX-WT protein stability.…”

Choreographing the Double Strand Break Response: Ubiquitin and SUMO Control of Nuclear Architecture

“…The current models posit that in addition to providing direct docking sites for protein substrates these ubiquitin chains also drive large scale chromatin changes. One such proposition is that 53BP1 binds to pre-existing H4-K20 dimethylated residues that are exposed locally by these DSB-specific modifications ( Huyen et al, 2004 ; Botuyan et al, 2006 ; Acs et al, 2011 ; Meerang et al, 2011 ; Mallette et al, 2012 ; Kocyłowski et al, 2015 ). More recent evidence demonstrates that 53BP1 is a specific reader of combinatorial histone modifications.…”