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
H2AX is a histone variant that differs from H2A at various amino acid residues along the entire protein and in its C-terminal extensions. H2AX is phosphorylated after the induction of DNA double-strand breaks (DSBs), and the phosphorylated H2AX (γ-H2AX) participates in focus formation at sites of DNA damage. After induction of DSBs, the MRN complex (M
AD50, and N
BS1) binds to broken DNA ends and recruits active ATM, ATR, and/or DNA protein kinase, resulting in the initial phosphorylation of H2AX (32
). MDC1 then associates with γ-H2AX and recruits additional activated ATM to the sites of DSBs (23
). This positive feedback loop leads to the expansion of the γ-H2AX region surrounding DSBs and provides docking sites for many DNA damage and repair proteins, including the MRN complex, 53BP1, and BRCA1 (5
). γ-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
). 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 γ-H2AX (34
). These findings raise the possibility that histone ubiquitination is also involved in the reorganization of chromatin in response to DSBs. To date, how the ubiquitination of histones is organized in the DNA damage response remains unknown.
We and other groups have shown that the histone acetyltransferase (HAT) TIP60/Esa1 participates in the DNA damage response as a protein complex (9
). For example, TIP60 induces histone H4 acetylation and the accumulation of repair molecules, including RAD51, at sites of DSBs with TRRAP in human cells (30
). In Saccharomyces cerevisiae
, the NuA4 complex, including Esa1, a yeast homologue of human TIP60, binds histone H4 through Arp4 to mediate the DSB-induced acetylation of H4 (9
). However, it is not yet known how histone acetylation by the TIP60 complex regulates chromatin organization immediately after the induction of DSBs in the human DNA repair response.
In addition to histone modifications, histone eviction/release and histone variant exchange can facilitate DNA repair by recruiting signaling and repair factors (12
). The exchange of core histones for a specific variant within nucleosomes can also alter chromatin structure in a temporally controlled manner (1
). For example, Drosophila melanogaster
DmTIP60 has been shown to acetylate phospho-H2A.v in vitro, resulting in its removal from chromatin due to exchange with an unmodified H2A.v (22
). In budding yeast, H2A (or phospho-H2A) is replaced with the H2A variant Htz1 by the histone exchange complex SWR1 (28
). The INO80 complex provokes chromatin reorganization following DNA damage; this reorganization includes the release of histones H2B and H3 at sites around DSBs, leading to the recruitment of RAD51 (29
). Furthermore, the histone variant H3.1 appears to be deposited at sites of UV damage by the chromatin assembly factor CAF-1 (36
). Although these studies indicate that histone eviction and variant exchange are important mechanisms for altering chromatin structure during the DNA damage response (35
), it is not clear how histone modifications engage in these chromatin reorganizations immediately following the induction of DSBs during DNA repair in human cells.
Here, we show that the TIP60 HAT complex interacts with H2AX immediately after exposure to ionizing irradiation (IR). Furthermore, DSBs facilitate the association of TIP60 with the ubiquitin-conjugating enzyme UBC13 (2
). The TIP60-UBC13 complex regulates the acetylation and ubiquitination of H2AX following the formation of DSBs. The DSB-induced acetylation of H2AX lysine 5 (K5) is required for this ubiquitination and occurs independently of the phosphorylation of H2AX. We also show that damage-induced acetylation and ubiquitination provoke the release of H2AX from chromatin immediately after the induction of DSBs. Because TIP60-UBC13 is required for the DSB-induced ubiquitination and release of H2AX, these findings provide the first evidence that human TIP60 promotes the acetylation-dependent ubiquitination of H2AX by UBC13, causing H2AX release from chromatin, which facilitates chromatin reorganization following DNA damage.