RESUMO
Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.
RESUMO
Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Histonas/metabolismo , Animais , Linhagem Celular , Dano ao DNA/efeitos da radiação , Reparo do DNA , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Lasers , Camundongos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , DNA de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA de Neoplasias/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Autoantígeno Ku , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Microscopia Confocal , Mutação , Ligação Proteica , Interferência de RNA , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.
Assuntos
Nitrilas/farmacologia , Nitrofuranos/farmacologia , Sulfonas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , NF-kappa B/antagonistas & inibidores , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Peptidilprolil Isomerase/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Histonas/química , Humanos , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Fosforilação , Ligação Proteica , Conformação Proteica , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteínas de Xenopus/metabolismoRESUMO
Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.
Assuntos
Dano ao DNA/efeitos dos fármacos , Histonas/química , Indanos/química , Complexo Repressor Polycomb 1/antagonistas & inibidores , Piridinas/química , Ubiquitina/metabolismo , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Microscopia de Fluorescência , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.
Assuntos
Dano ao DNA , Raios gama , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Humanos , Lasers , Lisina/metabolismo , Metilação/efeitos da radiação , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico/efeitos da radiação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.
Assuntos
Actinas/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Actinas/química , Antígenos Nucleares/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Autoantígeno Ku , PolimerizaçãoRESUMO
Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Sumoilação , Animais , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Células HEK293 , Humanos , Ligases , Lisina/metabolismo , Camundongos , Proteínas Nucleares/química , Poli(ADP-Ribose) Polimerases/metabolismo , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Proteínas Inibidoras de STAT Ativados/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Ubiquitina-Proteína LigasesRESUMO
Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/fisiologia , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Clonagem Molecular , Primers do DNA , Ativação Enzimática , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Humanos , Cinética , Proteína Homóloga a MRE11 , Camundongos , Camundongos Knockout , Neuroblastoma , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Poly(ADP-ribosyl)ation is a very early cellular response to DNA damage. Poly(ADP-ribose) (PAR) accumulation is transient since PAR is rapidly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). PARG may play a prominent role in DNA damage response and repair by removing PAR from modified proteins including PARP-1. Using living cells, we provide evidence that in response to DNA damage induced by gamma-irradiation the cytoplasmic 103 kDa PARG isoform translocates into the nucleus. We further observed that the nuclear GFP-hPARG110 enzyme relocalizes to the cytoplasm in response to DNA damage. Using different GFP-PARG fusion proteins specific for the nuclear and cytoplasmic forms, we demonstrate their dynamic distribution between cytoplasm and nucleoplasm and a high mobility of major PARG isoforms by fluorescence recovery after photobleaching (FRAP). The dynamic relocation of all PARG isoforms presented in this report reveals a novel biological mechanism by which PARG could be involved in DNA damage response.
Assuntos
Dano ao DNA , Glicosídeo Hidrolases/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Radiação Ionizante , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/efeitos da radiação , Difusão , Eletroforese em Gel de Poliacrilamida , Recuperação de Fluorescência Após Fotodegradação , Técnica Indireta de Fluorescência para Anticorpo , Glicosídeo Hidrolases/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Microscopia de Fluorescência , Neuroblastoma/patologia , Poli Adenosina Difosfato Ribose/genética , Frações Subcelulares/metabolismoRESUMO
DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C-XRCC3 or RAD51B-C-D-XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double-strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with gamma-H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S-G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo.