Genome stability is essential for prevention of undue cellular death and neoplasia. DNA damage caused by internal or external damaging agents is a major threat to the integrity of the cellular genome. The cellular defense system against this threat is the DNA damage response (DDR)—an elaborate signaling network that activates DNA repair and cell-cycle checkpoints and modulates many physiological pathways (Ciccia and Elledge, 2010
). The DDR serves as a barrier against cancer formation by activated oncogenes (Halazonetis et al., 2008
). Germline mutations abrogating critical relays in the DDR lead to genomic instability syndromes characterized by sensitivity to genotoxic stresses, tissue degeneration, and excessive cancers.
One of the most powerful activators of the DDR is the DNA double-strand break (DSB). This cytotoxic lesion is induced by ionizing radiation (IR), radiomimetic chemicals, and reactive oxygen species that accompany normal metabolism. DSBs are also formed during meiotic recombination and maturation of the antigen receptor genes (Hiom, 2010
). Eukaryotic cells repair DSBs via error-prone nonhomologous end-joining (NHEJ) or high-fidelity homologous recombination repair (HRR), which is preceded by DNA end resection (Holthausen et al., 2010
; Lieber, 2010
). The vast cellular response to DSBs affects cell-cycle progression, gene expression, protein synthesis, degradation and trafficking, and RNA processing.
The DSB response begins with recruitment of sensor proteins to the damaged sites; these proteins are involved in the initial recognition and processing of the damage and activation of the transducers of the DNA damage alarm (Ciccia and Elledge, 2010
). The primary transducer of the DSB response is the nuclear serine-threonine kinase ATM, which phosphorylates a plethora of effectors in various DDR pathways (Derheimer and Kastan, 2010
). Loss of ATM causes the genome instability syndrome ataxia-telangiectasia (A-T) (Lavin, 2008
A major question is how ATM controls a critical branch of the DDR: DSB repair. In laboratory cell lines, timely repair of about 10%–15% of IR-induced DSBs is ATM dependent (Riballo et al., 2004
). Like other DNA transactions, DNA repair should be coupled to chromatin reorganization. Chromatin organization is affected by protein-DNA and protein-protein interactions among core histones and nonhistone proteins and posttranslational modifications (PTMs) of these proteins. Dynamic changes in the rich repertoire of histone PTMs indeed accompany chromatin reorganization associated with DNA transactions (Murr, 2010
; Zhou et al., 2011
Here, we show that histone H2B monoubiquitylation, a highly dynamic and mobile histone mark known to be associated with transcription elongation, is induced in human cells after DSB induction in an ATM-dependent manner. The responsible E3 ubiquitin ligase is a heterodimer of the RING-finger proteins RNF20 and RNF40 (Kim et al., 2005
; Zhu et al., 2005
). Human RNF20 and RNF40 are orthologs of the budding yeast protein Bre1, which together with the ubiquitin-conjugating enzyme Rad6, monoubiquitylates the yeast histone H2B on lysine 123 at sites of transcription elongation (Kao et al., 2004
). This histone modification was recently shown to interfere with the compaction of the basic 30 nm chromatin fiber (Fierz et al., 2011
). Notably, in yeast and mammalian cells, H2B monoubiquitylation at transcribed chromatin is required for subsequent histone H3 methylations on Lys4 and Lys79 (Dover et al., 2002
; Kim et al., 2009
; Lee et al., 2007
; Schneider et al., 2005
We find that, similar to transcription-associated H2B monoubiquitylation, the induction of this histone modification after DNA damage is RNF20-RNF40 dependent. Furthermore, upon DNA damage a fraction of RNF20-RNF40 is recruited to DSB sites and undergoes ATM-mediated phosphorylation. ATM’s activity and the presence of its phosphorylation sites on RNF20-RNF40 are required for damage-induced H2B monoubiquitylation. This process is not required for the recruitment of the damage sensors in the very early stage of the DDR, but is essential for timely accumulation of NHEJ and HRR proteins at DSB sites and subsequent optimal repair via both pathways. Collectively, our data and previous data suggest a two-stage process of chromatin relaxation at DSB sites that is essential for timely DSB repair.