DNA Damage-Dependent Acetylation and Ubiquitination of H2AX Enhances Chromatin Dynamics

Abstract

Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.Chromatin reorganization by histone modification and mobilization plays a crucial role in DNA metabolism, including replication, transcription, and repair. It appears that histone modification and mobilization can reorganize chromatin to allow DNA repair machinery to access damaged chromosomal DNA (11,29,52,56,57).H2AX is a histone variant that differs from H2A at various amino acid residues along the entire protein and in its Cterminal extensions. H2AX is phosphorylated after the induction of DNA double-strand breaks (DSBs), and the phosphorylated H2AX (␥-H2AX) participates in focus formation at sites of DNA damage. After induction of DSBs, the MRN complex (MRE11, RAD50, and NBS1) binds to broken DNA ends and recruits active ATM, ATR, and/or DNA protein kinase, resulting in the initial phosphorylation of H2AX (32,38,40). MDC1 then associates with ␥-H2AX and recruits additional activated ATM to the sites of DSBs (23,46). This positive feedback loop leads to the expansion of the ␥-H2AX region surrounding DSBs and provides docking sites for many DNA damage and repair proteins, including the MRN complex, 53BP1, and BRCA1 (5, 6, 46). ␥-H2AX plays a role in the accumulation but not in the initial recruitment of repair factors such as the MRN complex, 53BP1, and BRCA1 (10, 63). Therefore, modifications of H2AX other than phosphorylation could play a role in the initial step of the DNA damage response.Until recently, the biological significance of ubiquitination in the DNA damage response has been unclear. H2B ubiquitination regulates the damage checkpoint response (15). H2A is ubiquitinated during the response to UV-induced DNA damage (8). UV-induced DNA damage also causes the ubiquitination of histones H3 and H4, resulting in their release from chromatin (60). Interestingly, ubiquitin-conjugated proteins appear to be accumulated at sites of DSBs, forming nuclear foci like ␥-H2AX (34). These findings raise the possibility that histone ubiquitination is also involved in the reorganization of chromatin in response to D...

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…This finding remains contentious -they themselves found that, despite the reduced chromatin partitioning of MDC1 upon NCL depletion, immunofluorescent localization of MDC1 to DSB foci was unaffected, while Goldstein and colleagues' ChIP-based assay found no reduction in the recruitment of MDC1 to a defined DSB site in the absence of NCL (Goldstein et al 2013). It is known, however, that RNF8/RNF168-dependent H2A/H2B ubiquitination is an important activating step in histone eviction both following DNA damage and during spermiogenesis, in which histone octamers are globally evicted from DNA and replaced by protamines to facilitate greater DNA compaction; such observations suggest that regulation of MDC1 and its partners by NCL could conceivably play a role in NCL's histone-chaperone activity at DSBs (Ikura et al 2007, Lu et al 2010, Rathke et al 2014. Collectively, these data argue for a model in which NCL recruitment to DSBs via the MRN complex facilitates the proximal nucleosome destabilization by chromatin remodeling complexes, possibly through the actions of MDC1 and its targets RNF8/RNF168, and ultimately facilitates NHEJ-and HR-mediated DSB resolution (Goldstein et al 2013).…”

Chromatin modification and NBS1: their relationship in DNA double-strand break repair

“…Downs and her colleagues also reported that Arp4, a subunit of the NuA4 HAT complex, is recruited to HO endonuclease-induced DSB sites and interacts with γ-H2A directly in yeast (Downs et al, 2004). In mammalian cells, TIP60, the HAT component of the mammalian homolog of the NuA4 HAT complex, can be recruited to a DSB site by a physical interaction with the MRN complex and γ-H2AX (Ikura et al, 2000(Ikura et al, , 2007Chailleux et al, 2010). We also reported that TIP60 could acetylate histone H2A, and that this acetylation might be essential for DNA damage-induced focus formation and HR repair (Kobayashi et al, 2010).…”

Chromatin dynamics coupled to DNA repair

“…In addition, it is possible that acetylated H4 tail is specifically recognized by chromatin remodeling complexes or other effectors that can induce γH2AX eviction and relaxation of the chromatin fiber, thus favoring the recruitment of DNA repair proteins. In accordance with the latter possibility, Ikura et al 82 showed Tip60 to be required for the mobilization of γH2AX in the first 5 min after DSB induction with ionizing radiation. Tip60 acetylates H2AX on K5, which promotes the UBC13-mediated polyubiquitination of H2AX on K119 before releasing H2AX from chromatin.…”

“…Tip60 acetylates H2AX on K5, which promotes the UBC13-mediated polyubiquitination of H2AX on K119 before releasing H2AX from chromatin. 82 However, the γH2AX acetylation was not observed after UV radiation. 85 This difference could be related to the various agents used to induce DNA damage.…”

“…78,84 In mammals, TRRAP/TIP60 is recruited to DSB, where it acetylates H4 and H2AX. 81,82 In fact, the generation of a single DSB induces hyperacetylation of histone H4 in the surrounding chromatin. Deletion of TRRAP in mouse cells impairs Tip60 recruitment and H4 acetylation and reduces the recruitment of 53BP1, Rad51 and BRCA1 at DSB sites.…”