The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

Induction of cancerous stem cells during embryonic stem cell differentiation.

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.

Artemis-dependent DNA double-strand break formation at stalled replication forks.

Stalled replication forks undergo DNA double-strand breaks (DSBs) under certain conditions. However, the precise mechanism underlying DSB induction and the cellular response to persistent replication fork stalling are not fully understood. Here we show that, in response to hydroxyurea exposure, DSBs are generated in an Artemis nuclease-dependent manner following prolonged stalling with subsequent activation of the ataxia-telangiectasia mutated (ATM) signaling pathway. The kinase activity of the catalytic subunit of the DNA-dependent protein kinase, a prerequisite for stimulation of the endonuclease activity of Artemis, is also required for DSB generation and subsequent ATM activation. Our findings indicate a novel function of Artemis as a molecular switch that converts stalled replication forks harboring single-stranded gap DNA lesions into DSBs, thereby activating the ATM signaling pathway following prolonged replication fork stalling.

The quiescent cellular state is Arf/p53-dependent and associated with H2AX downregulation and genome stability.

Cancer is a disease associated with genomic instability and mutations. Excluding some tumors with specific chromosomal translocations, most cancers that develop at an advanced age are characterized by either chromosomal or microsatellite instability. However, it is still unclear how genomic instability and mutations are generated during the process of cellular transformation and how the development of genomic instability contributes to cellular transformation. Recent studies of cellular regulation and tetraploidy development have provided insights into the factors triggering cellular transformation and the regulatory mechanisms that protect chromosomes from genomic instability.

Transcription-dependent activation of ataxia telangiectasia mutated prevents DNA-dependent protein kinase-mediated cell death in response to topoisomerase I poison.

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

DPPA4 modulates chromatin structure via association with DNA and core histone H3 in mouse embryonic stem cells.

Developmental pluripotency associated 4 (DPPA4) is one of the uncharacterized genes that is highly expressed in embryonic stem (ES) cells. DPPA4 is associated with active chromatin and involved in the pluripotency of mouse ES cells. However, the biological function of DPPA4 remains poorly understood. In this study, we performed fluorescence recovery after photobleaching (FRAP) analysis to examine the dynamics of DPPA4 in ES cells. FRAP analysis showed that the mobility of DPPA4 is similar to that of histone H1. In addition, biochemical analysis with purified proteins and immunoprecipitation analysis showed that DPPA4 directly binds to both DNA and core histone H3. The analysis using truncated proteins indicated that DPPA4 is associated with DNA via the N-terminal region and histone H3 via the C-terminal region. In vitro assembled chromatin showed resistance to micrococcal nuclease (MNase) digestion in the presence of DPPA4. Moreover, MNase assay and FRAP analysis with the truncated proteins implies that DPPA4 binding to both DNA and histone H3 is necessary for the chromatin structure resistant to MNase and for the proper localization of DPPA4 in ES cell nuclei. These results suggest that DPPA4 modulates the chromatin structure in association with DNA and histone H3 in ES cells.

RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair.

The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

Role of Parp-1 in suppressing spontaneous deletion mutation in the liver and brain of mice at adolescence and advanced age.

Poly(ADP-ribose) polymerase-1 knockout (Parp-1(-/-)) mice show increased frequency of spontaneous liver tumors compared to wild-type mice after aging. To understand the impact of Parp-1 deficiency on mutations during aging, in this study, we analyzed spontaneous mutations in Parp-1(-/-) aged mice. Parp-1(-/-) mice showed tendencies of higher mutation frequencies of the red/gam genes at 18 months of age, compared to Parp-1(+/+) mice, in the liver and brain. Complex-type deletions, accompanying small insertion were observed only in Parp-1(-/-) mice in the liver and brain. Further analysis in the liver showed that the frequency of single base deletion mutations at non-repeat or short repeat sequences was 5.8-fold higher in Parp-1(-/-) than in Parp-1(+/+) mice (p<0.05). A 3.2-fold higher tendency of the deletion frequency of two bases or more was observed in Parp-1(-/-) mice compared to Parp-1(+/+) mice (p=0.084). These results support the model that Parp-1 is involved in suppressing imprecise repair of endogenous DNA damage leading to deletion mutation during aging. The mutation frequencies of the gpt gene in the brain were found to be 3-fold lower in Parp-1(-/-) than in Parp-1(+/+) mice at 4 months of age (p<0.01), implying that Parp-1 may be positively involved in imprecise DNA repair in the brain. On the other hand, the frequencies of gpt mutation showed an increase at 18 months of age in the Parp-1(-/-) (p<0.05) but not in Parp-1(+/+) brains, suggesting that Parp-1 deficiency causes an increase of point mutations in the brain by aging.

Involvement of CCAAT/enhancer binding protein-beta (C/EBPbeta) in epigenetic regulation of mouse methionine adenosyltransferase 1A gene expression.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the main methyl donor in cellular transmethylation reactions and the aminopropyl moiety in polyamine biosynthesis. In mammals, two different genes, MAT1A and MAT2A, encode catalytic polypeptides of liver-specific MAT I/III and ubiquitous MAT II, respectively. Reverse transcription-polymerase chain reaction showed that MAT1A gene expression was at a detectable level in embryonic day 14 mouse fetal liver and subsequently increased. Bisulfite genomic sequencing indicated that the methylation status of 10CpG sites in the MAT1A promoter proximal region was appreciably correlated with the gene expression in mouse developing liver and in adult hepatic cells; hepatic stellate cells and hepatocytes. When mouse hepatoma-derived Hepa-1 cells showing extremely low expression of MAT1A gene were treated with 5-aza-2'-deoxycytidine and trichostatin A, MAT1A gene expression was enhanced. In addition, in vitro methylation of the MAT1A promoter region suppressed the MAT1A promoter activity in reporter assay. Next, we performed electrophoretic mobility shift assay and found that the transcriptional factor CCAAT/enhancer binding protein-beta (C/EBPbeta) specifically binds to a putative binding site of C/EBPbeta in the MAT1A promoter. Suppression of C/EBPbeta expression by short hairpin RNA decreased the MAT1A promoter activity and MAT1A gene expression, and inhibition of C/EBPbeta binding to MAT1A by site-directed mutagenesis also showed similar results. Western blot analysis and chromatin immunoprecipitation assay indicated that C/EBPbeta binding is dependent on DNA methylation status. Based on these findings, we conclude that C/EBPbeta plays an important role in epigenetic regulation of the mature hepatic gene MAT1A.

Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-alpha and -beta.

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.

Distamycin A enhances the cytotoxicity of duocarmycin A and suppresses duocarmycin A-induced apoptosis in human lung carcinoma cells.

Duocarmycin A (Duo), which is one of well-known antitumor antibiotics, efficiently alkylates adenine N3 at the 3' end of AT-rich sequences in the DNA. The addition of a minor groove binder, distamycin A (Dist), not only accerelates the reactivity of Duo with oligonucleotide duplex but also switches the DNA-alkylation site to guanine in GC-rich sequences. Here we examined cytotoxic effect of Duo in the coexistence of Dist using human lung carcinoma (HLC-2) cells. The cytotoxicity of Duo to HLC-2 cells was enhanced 10 times by the addition of 0.5microg/ml Dist, which was much lower than the IC(50) value of 16microg/ml. Addition of Duo alone to HLC-2 cells resulted in typically apoptotic changes, including chromatin condensation, sub-G1 accumulation in DNA histogram pattern, and decrease in procaspase-3 and 9 levels. Interestingly, these apoptotic characteristics in Duo-treated cells were suppressed by the addition of 0.5microg/ml Dist, and the G2/M population in the cell cycle progression of HLC-2 cells was largely unchanged in the coexistence of Dist along with the extremely low accumulation of p53 and higher induction of p21. In contrast, the treatment of HLC-2 cells with Dist (16microg/ml) alone was observed to induce the accumulation of p53 and cell cycle arrest at the G1 phase. These results indicate that Dist suppresses apoptosis induced by Duo as well as enhances Duo-induced cytotoxicity in living cells, and may contribute to chemotherapy for tumors resistant to inducing apoptotic cell death.

The intrinsic structure of glucose transporter isoforms Glut1 and Glut3 regulates their differential distribution to detergent-resistant membrane domains in nonpolarized mammalian cells.

The hexose transporter family, which mediates facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. We previously demonstrated that glucose transporter 1 is organized into a raft-like detergent-resistant membrane domain but that glucose transporter 3 distributes to fluid membrane domains in nonpolarized mammalian cells. In this study, we further examined the structural basis responsible for the distribution by using a series of chimeric constructs. Glucose transporter 1 and glucose transporter 3 with a FLAG-tagged N-terminus were expressed in detergent-resistant membranes and non-detergent-resistant membranes of CHO-K1 cells, respectively. Replacement of either the C-terminal or N-terminal cytosolic portion of FLAG-tagged glucose transporter 1 and glucose transporter 3 did not affect the membrane distribution. However, a critical sorting signal may exist within the N-terminal half of the isoforms without affecting transport activity and its inhibition by cytochalasin B. Further shortening of these regions altered the critical distribution, suggesting that a large proportion or several parts of the intrinsic structure, including the N-terminus of each isoform, are involved in the regulation.

Hepatic progenitor cells in the mouse extrahepatic bile duct after a bile duct ligation.

The intrahepatic bile duct has been suggested to be a source of hepatic progenitor cells in the severely damaged liver. In contrast, little attention has been paid to the question of whether hepatic progenitor cells exist in the extrahepatic bile duct (EHBD). In the present study, we examined the phenotypic changes of the mouse EHBD following bile duct ligation. After bile duct ligation, the number of c-Kit-positive epithelial cells increased in the EHBD. The ligated EHBD expressed mRNA for hepatic progenitor cell markers, including c-Kit and Thy-1. Hepatocyte markers such as albumin and cytochrome P450 7a1 were also transiently detected in the EHBD after a bile duct ligation. In a culture of EHBD cells, we detected hepatic progenitor cells that were positive for both staining with anti-albumin antibodies and Dolichos biflorus agglutinin, a biliary epithelial cell-specific lectin. Furthermore, hepatic progenitor cells positive for both c-Kit and albumin were found in the cultured EHBD population. Additionally EHBD-derived hepatocyte-like cells were also observed in the culture. A transplantation study revealed that EHBD cells integrate into the parenchyma and are albumin positive. These data suggest that hepatic progenitor cells emerge in the EHBD following bile duct ligation, that subsequently give rise to hepatocyte-like cells. We also observed that the gall bladder transiently expressed hepatocyte markers after bile duct ligation. Our results suggest a potential of the EHBD and gall bladder as useful transplantable sources for liver injury.

Twist relates to tubular epithelial-mesenchymal transition and interstitial fibrogenesis in the obstructed kidney.

Epithelial-mesenchymal transition (EMT) is a critical step in renal fibrosis. It has been recently reported that a transcription factor, Twist, plays a pivotal role in metastasis of breast tumors by inducing EMT. In this study, we examined whether Twist relates to renal fibrogenesis including EMT of tubular epithelia, evaluating Twist expression level in the unilateral ureteral obstruction (UUO) model. Kidneys of mice subjected to UUO were harvested 1, 3, 7, and 10 days after obstruction. Compared with control kidneys, Twist mRNA-level significantly increased 3 days after UUO (UUO day 3 kidney) and further augmented until 10 days after UUO. Twist expression increased in tubular epithelia of the dilated tubules and the expanded interstitial areas of UUO kidneys, where cell-proliferating appearances were frequently found in a time-dependent manner. Although a part of tubular cells in whole nephron segment were immunopositive for Twist in UUO day 7 kidneys, tubular epithelia downstream of nephron more frequently expressed Twist than upstream of nephron. In UUO day 7 kidneys, some tubular epithelia were confirmed to coexpress Twist and fibroblast-specific protein-1, a marker for EMT, indicating that Twist is involved in tubular EMT under pathological state. Twist was expressed also in a number of alpha-smooth muscle actin-positive myofibroblasts located in the expanded interstitial area of UUO kidneys. From these findings, the present investigation suggests that Twist is associated with tubular EMT, proliferation of myofibroblasts, and subsequent renal fibrosis in obstructed kidneys.

Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1-/- than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

Coprinus cinereus DNA ligase I during meiotic development.

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source.

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.

Development of cancer-initiating cells and immortalized cells with genomic instability.

Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations and epigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells (CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A (ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2AX to levels representative of growth arrest in normal cells.

ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation.

In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.