We sought to determine whether the ubiquitous, preexisting basal levels of H4K16 acetylation have a role in DDR and what impact H4K16ac's absence may have on DNA DSB repair by NHEJ or HR pathways. Our results provide strong evidence that preexisting H4K16ac does facilitate DNA damage recognition (Fig. ). Based on the established role of this modification in chromatin organization, our observations are consistent with the model wherein basal levels of H4K16ac maintain a chromatin structure conducive for efficient DNA damage repair. This model (Fig. ) is based on the fact that specifically altering the levels of H4K16ac by various approaches directly affected IR-induced γ-H2AX focus formation: (i) MOF depletion reduced H4K16ac levels and delayed/abrogated IR-induced focus formation of γ-H2AX, MDC1, 53BP1, Rad51, and hSSB1; (ii) depletion of Tip60, another HAT, had no impact on H4K16ac levels and no influence on γ-H2AX focus formation; (iii) depletion of Tip60 did not enhance the delay in the IR-induced γ-H2AX focus formation in cells depleted for hMOF; (iv) cells expressing HAT-dead MOF had a delay in the appearance of IR-induced γ-H2AX focus formation; (v) treatment of cells with TSA enhanced H4K16ac levels and reversed the delay in γ-H2AX focus formation upon depletion of hMOF; (vi) MOF depletion had a minimal effect on the appearance of IR-induced γ-H2AX foci in SirT2−/− cells as compared to SirT2+/+ cells; and (vii) MOF depletion along with ATM inactivation increased the delay in the formation of IR-induced γ-H2AX foci.
FIG. 7. MOF and H4K16ac influence DDR at multiple stages of DNA DSB repair pathways. MOF is a major HAT for H4K16ac, and its levels determine IR-induced repairosome formation. MOF interacts with DNA-PKcs and also localizes on the synaptonemal complex (SCP3) of (more ...)
Our comprehensive analysis of the correlation between the chromatin modifier MOF, modified histone H4 residue K16ac, and IR-induced DDR strongly supports the argument that acetylation of histone H4K16 strongly influences DDR (Fig. ). The role of H4K16ac in DDR is further supported by the fact that the acetylation at the N-terminal tails of histones H3 and H4 correlates with the establishment of an open euchromatin conformation that is transcriptionally active. Conversely, hypoacetylation of H3 and H4 tails is associated with heterochromatin (36
). Our results argue that the two different forms of chromatin could differentially influence generation of DDR-associated signaling. Histone acetylation impacts chromatin structure through the neutralization of the positive charges on lysine residues, altering intra- and internucleosomal interactions of the chromatin fiber and thus facilitating decondensation and enhancing nucleosomal DNA accessibility (6
). It is well established that histone acetylation is recognized by transcriptional factors or ATP-dependent remodeling activities (91
), but since acetylated K16 (H4K16ac) is present in ~60% of the total H4 molecules of mammalian cells (84
), it is likely that it also has a specific structural role in chromatin-based processes such as DDR. Shogren-Knaak and coworkers have demonstrated the structural significance of histone H4 at lysine 16 (H4K16ac) as its incorporation into nucleosomal arrays abrogates the formation of compact 30-nm-like fibers (74
). The adaptor protein 14-3-3 binds the phosphorylated nucleosome and recruits MOF, which triggers the acetylation of histone H4 at lysine 16 (94
). Nucleosomes with H4K16ac in the fiber have a decreased ability to form cross-fiber interactions (74
), and thus the chromatin remains in an open state, conducive to DDR. This assumption is consistent with the fact that H4K16ac levels peak in S phase (91
), the cell cycle phase most efficient for DNA DSB repair and which has less IR-induced cell killing than during the G1
phases of the cell cycle (55
Histone acetyltransferases exist as components of multisubunit protein complexes involved in different processes such as transcription activation, gene silencing, and cell cycle progression as well as DNA damage repair (12
). Such complexes have been reported to contain ATM-related proteins such as TRRAP, which is found in human Tip60 and PCAF complexes (3
). ATM is implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell cycle control, and telomere maintenance (50
The effect of MOF on ATM activation is by an indirect mechanism, and inactivation of ATM or p53 does not rescue lethality due to MOF deletion (24
). In addition, our current studies reveal that depletion of MOF does not influence the levels of proteins known to be involved in DDR, suggesting that MOF-dependent H4K16ac primarily affects DDR through a direct impact on chromatin structure (Fig. ). Given the significance of H4K16ac, its absence could result in reduced transmission of signals generated from DNA damage sites, and this would delay the timely course of repair. Furthermore, analysis of H4K16ac levels at the site of I-SceI-induced DSB in MOF-depleted cells revealed a direct correlation between loss of H4K16ac and abrogation of γ-H2AX focus appearance.
In addition to histone kinases, several studies have revealed the role of HAT complexes in DNA DSB repair. It has been reported that HAT complexes act in concert with the ATP-dependent SWI/SNF and RSC (which remodels the structure of chromatin)-containing chromatin-remodeling complexes to facilitate DNA repair (58
). Cells expressing catalytically inactive TIP60 have been found to have impaired DNA DSB repair (30
). TIP60 with its cofactor TRRAP directly binds to chromatin near DNA DSBs, and depletion of TRAPP impairs DNA damage-induced H4 acetylation, which results in defective DSB repair by HR (45
). Similar results were observed with NuA4, a yeast homolog of TIP60, following induction of DSB by HO endonuclease (17
). NuA4 binds directly to sites of DNA DSBs concomitantly with the appearance of γ-H2AX (17
Our analysis revealed that MOF influences the formation of DNA DSB repair-associated foci, but its global association with chromatin increases postirradiation, as determined by ChIP assay (Fig. ). Furthermore, MOF retention on chromatin also increases postirradiation, suggesting that MOF itself has a critical role in the later steps of DNA DSB repair, which is evidenced by the fact that cells expressing mutant MOF had higher residual DNA DSBs and chromosome aberrations (25
). Our previous studies have revealed that expression of mutant hMOF induced neither a significant increase in G1
-phase cells nor a decrease in S-phase entry or accumulation in G2
phase after IR exposure compared to control cells (25
). However, cells expressing a HAT-dead mutant of hMOF had a higher frequency of IR-induced chromosomal aberrations, suggesting that hMOF does influence DNA DSB repair (25
Further evidence for the role of MOF in DNA DSB repair is provided by the following observations: (i) MOF forms a complex with DNA-PKcs, and (ii) MOF localizes on the synaptonemal complexes in spermatocytes. Depletion of MOF resulted in abrogation of IR-induced ATM-dependent phosphorylation of DNA-PKcs. These observations are consistent with the model proposed earlier that MOF regulates the ATM function through the status of chromatin (Fig. ). Furthermore, depletion of MOF delays the accumulation of DNA-PKcs postirradiation. Delay in the accumulation could be due to following reasons: (i) loss of ATM-dependent damage-induced DNA-PKcs phosphorylation; (ii) change in chromatin structure due to loss of H4K16ac; and (iii) decreased association of MOF with DNA postirradiation, preventing chromatin alterations conducive for DSB repair.
hMOF functions upstream of ATM (25
), possibly sensing DNA damage-induced chromatin changes with subsequent signaling to ATM effectors. In contrast, Tip60 has been reported to activate ATM by acetylation of its lysine residue, and so ATM activation could be independent of chromatin modifications (78
). Overall, histone acetylation appears to both unwind chromatin and create a permissive platform that facilitates recruitment of remodeling complexes. The INO80 complex, including the INO80 member of the SWI/SNF family, has long been known to regulate transcription at RNA polymerase II (Pol II) promoters through chromatin remodeling. More recently, it was observed that INO80 is recruited to γ-H2AX near DNA DSBs, and yeast mutants of INO80 are hypersensitive to damaging agents and HO endonuclease, providing one of the first examples of SWI/SNF ATPase participation in DNA repair (44
). Interestingly, the actin-related protein Arp4 in yeast is present in both the NuA4-HAT complex and the INO80-SWR1 complex, further suggesting concerted action of histone-modifying and chromatin-remodeling activities in the DSB response (17
). The exact role of INO80 has yet to be clearly defined. TRRAP-TIP60 activity may be limited to local chromatin unwinding since chromatin relaxation alone is sufficient to rescue the defects caused by TRRAP deficiency (45
). Since the role of MOF through alteration of H4K16ac levels is upstream in DDR and MOF forms a complex with DNA-PKcs, essential for NHEJ, our studies also found that MOF is required for the repair of DNA DSB by the HR pathway. This is supported by the observations that depletion of MOF results in the abrogation of Rad51 focus formation (Fig. ) and reduced frequency of sister chromatid exchange formation (see Fig. S10 in the supplemental material). Further evidence about the role of MOF comes from the following observations: (i) depletion of MOF results in reduced frequency of I-SceI-induced DSB repair, and (ii) MOF is localized on the synapetonemal complex during the process of meiotic recombination.
In contrast to yeast, which has only two copies of histone H4, studying the direct role of lysine 16 of histone H4 in mammalian cell systems is more difficult due to the presence of multiple copies of the H4 gene. Moreover, the yeast and mammalian systems are substantially different, since deletion of Sas2, the main H4K16 acetyltransferase in yeast, does not result in cellular lethality, whereas deletion of MOF not only results in embryonic lethality but also has a cytostatic effect in many human and mouse cell lines examined (24
). Although loss of acetylation at H4K16 does influence transcription in both yeast (14
) and mammalian (32
) cells, it is important to note that chromatin modifications common to yeast and mammalian systems do not always lead to similar DDR phenotypes. Acetylation of H4K16, for example, results in loss of histone H4 in yeast but does not result in loss of histone H4 in mammalian cells. This could be due to the fact that mammalian cells have a much more complex chromatin organization, so that H4K16ac could be playing a more critical role in DDR, as is further supported by the present work.
In summary, these data indicate that H4K16ac (histone code) constitutes one of the main early signals for generation of efficient DDR, which influences both of the pathways of DNA DSB repair. These results are consistent with the finding that chromatin alterations due to DNA DSB trigger ATM phosphorylation in mammalian cells, which is abrogated when cells are depleted of MOF. Abrogation of both DNA DSB repair pathways at several stages in cells depleted of MOF and the presence of MOF in the synaptonemal complex are further strong evidence of the role of MOF in DDR. Since MOF is indispensable for both NHEJ and HR DNA DSB repair pathways, H4K16ac (a histone code) must provide a permissive chromatin environment critical for DDR and subsequent mammalian cell survival. Further studies should reveal whether the H4K16ac levels of any particular region of the genome are critical for DDR.