H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes.

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability.

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Rad52 inactivation is synthetically lethal with BRCA2 deficiency.

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.

Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes.

Ataxia-telangiectasia mutated (ATM)-deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor alpha/delta locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient alphabeta T cells with translocations involving the T cell receptor alpha/delta locus is directly related to the number of T cell receptor alpha rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment.

ATM stabilizes DNA double-strand-break complexes during V(D)J recombination.

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

Isolation and Culture of Periosteum-Derived Progenitor Cells from Mice.

This chapter describes the methods of isolation of mouse periosteal progenitor cells. There are three basic methods utilized. The bone grafting method was developed utilizing the fracture healing process to expand the progenitor populations. Bone capping methods requires enzymatic digestion and purification of cells from the native periosteum, while the Egression/Explant method requires the least manipulation with placement of cortical bone fragments with attached periosteum in a culture dish. Various cell surface antibodies have been employed over the years to characterize periosteum derived progenitor cells, but the most consistent minimal criteria was recommended by the International Society for Cellular Therapy. Confirmation of the multipotent status of these isolated cells can be achieved by differentiation into the three basic mesodermal lineages in vitro.

Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

A role for the HOXB7 homeodomain protein in DNA repair.

Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.

Involvement of human MOF in ATM function.

We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasia-mutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant DeltahMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.

Molecular and epigenetic regulatory mechanisms of normal stem cell radiosensitivity.

Ionizing radiation (IR) therapy is a major cancer treatment modality and an indispensable auxiliary treatment for primary and metastatic cancers, but invariably results in debilitating organ dysfunctions. IR-induced depletion of neural stem/progenitor cells in the subgranular zone of the dentate gyrus in the hippocampus where neurogenesis occurs is considered largely responsible for deficiencies such as learning, memory, and spatial information processing in patients subjected to cranial irradiation. Similarly, IR therapy-induced intestinal injuries such as diarrhea and malabsorption are common side effects in patients with gastrointestinal tumors and are believed to be caused by intestinal stem cell drop out. Hematopoietic stem cell transplantation is currently used to reinstate blood production in leukemia patients and pre-clinical treatments show promising results in other organs such as the skin and kidney, but ethical issues and logistic problems make this route difficult to follow. An alternative way to restore the injured tissue is to preserve the stem cell pool located in that specific tissue/organ niche, but stem cell response to ionizing radiation is inadequately understood at the molecular mechanistic level. Although embryonic and fetal hypersensity to IR has been very well known for many decades, research on embryonic stem cell models in culture concerning molecular mechanisms have been largely inconclusive and often in contradiction of the in vivo observations. This review will summarize the latest discoveries on stem cell radiosensitivity, highlighting the possible molecular and epigenetic mechanism(s) involved in DNA damage response and programmed cell death after ionizing radiation therapy specific to normal stem cells. Finally, we will analyze the possible contribution of stem cell-specific chromatin's epigenetic constitution in promoting normal stem cell radiosensitivity.

Transient PP2A inhibition alleviates normal tissue stem cell susceptibility to cell death during radiotherapy.

Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice.

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.

Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation.

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.

Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53.

The exact duplication of chromosomal DNA during each cell cycle ensures the correct inheritance of genetic material from mother to daughter cells. In eukaryotic cells, DNA replication can occur only when the origin of DNA replication is accurately marked by a group of proteins termed licensing proteins. One such protein is Cdt1, which is recruited first to the origin of DNA replication followed by cell division cycle 6 (Cdc6) and mini-chromosome maintenance proteins (Mcms). We previously reported that NIH3T3 cells overexpressing Cdt1 readily formed tumors in mice. To further investigate its oncogenic mechanism, we generated transgenic mice expressing Cdt1 in thymocytes. Our studies demonstrated that T-cell-directed Cdt1 transgenic mice showed normal T-cell development. However, such transgenic mice developed thymic lymphoblastic lymphoma when crossed with p53 null mice. Furthermore, tumor cells derived from NIH3T3 cells overexpressing Cdt1 displayed numerical and structural chromosomal aberrations in the form of ploidy, double minutes, translocation, inversion, chromosome end-to-end fusion and robertsonian mutation. Collectively, our studies suggest that Cdt1 overexpression most likely contributes to tumorigenecity by causing genomic instability.

Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells.

Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.

Unique epigenetic influence of H2AX phosphorylation and H3K56 acetylation on normal stem cell radioresponses.

Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non-stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation-induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.

hTERT associates with human telomeres and enhances genomic stability and DNA repair.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

Telomere stability correlates with longevity of human beings exposed to ionizing radiations.

Normal somatic cells have a finite number of divisions, a limited capacity to proliferate. Human telomeres contain TTAGGG repeats which are considered a molecular clock marker. The gradual and progressive telomere shortening at each replicative cycle is associated, through the activation of pRB and p53 pathways and genomic instability, to the replicative senescence, a non-dividing state and widespread cell death. There is no information available about telomere status in individuals who live long and have been exposed to ionizing radiations (IR). To determine the telomere stability, we examined telomeres at metaphase, G2-type chromosome aberrations after IR treatment and karyotypic analysis of 15 individuals. Three individuals were above the age of 80 years and 1 among the 3 was estimated to have received more than 10 Gy of occupational exposure about 30 years back. The other 12 were cancer patients that had received more than 50 Gy of gamma-radiation for therapeutic purposes. No telomere instability or defective G2 chromosome repair was found in 3 individuals above the age of 80 years. Whereas, 3 out of 7 prostate and 1 out of 5 breast cancer patients showed higher G2-type chromosome damage as well as a high frequency of telomeric association (also known as chromosome end associations) along with frequent loss of telomeres. Present studies demonstrate that telomere stability along with normal G2 chromosome repair correlates with the longevity of human beings, whereas defective G2 chromosome repair and telomere instability correlate with the radiotherapy related late toxicity.

Genome-wide distribution of histone H4 Lysine 16 acetylation sites and their relationship to gene expression.

BACKGROUND: Histone post-translational modifications are critical determinants of chromatin structure and function, impacting multiple biological processes including DNA transcription, replication, and repair. The post-translational acetylation of histone H4 at lysine 16 (H4K16ac) was initially identified in association with dosage compensation of the Drosophila male X chromosome. However, in mammalian cells, H4K16ac is not associated with dosage compensation and the genomic distribution of H4K16ac is not precisely known. Therefore, we have mapped the genome-wide H4K16ac distribution in human cells. RESULTS: We performed H4K16ac chromatin immunoprecipitation from human embryonic kidney 293 (HEK293) cells followed by hybridization to whole-genome tiling arrays and identified 25,893 DNA regions (false discovery rate <0.005) with average length of 692 nucleotides. Interestingly, although a majority of H4K16ac sites localized within genes, only a relatively small fraction (~10%) was found near promoters, in contrast to the distribution of the acetyltransferase, MOF, responsible for acetylation at K16 of H4. Using differential gene expression profiling data, 73 genes (> ±1.5-fold) were identified as potential H4K16ac-regulated genes. Seventeen transcription factor-binding sites were significantly associated with H4K16ac occupancy (p < 0.0005). In addition, a consensus 12-nucleotide guanine-rich sequence motif was identified in more than 55% of the H4K16ac peaks. CONCLUSIONS: The results suggest that H4K16 acetylation has a limited effect on transcription regulation in HEK293 cells, whereas H4K16ac has been demonstrated to have critical roles in regulating transcription in mouse embryonic stem cells. Thus, H4K16ac-dependent transcription regulation is likely a cell type specific process.

MOF and histone H4 acetylation at lysine 16 are critical for DNA damage response and double-strand break repair.

The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.