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1.
Genes Dev ; 30(2): 149-63, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26744420

RESUMO

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.


Assuntos
Proteínas de Transporte/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Proteínas Nucleares/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/imunologia , Estrutura Molecular , Estrutura Terciária de Proteína , Transporte Proteico
2.
Proc Natl Acad Sci U S A ; 114(44): E9328-E9337, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29078319

RESUMO

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation.


Assuntos
Subpopulações de Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Cromatina/imunologia , Imunidade Humoral/imunologia , Proteínas Nucleares/imunologia , Animais , Medula Óssea/imunologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Proliferação de Células/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Linfocitária/imunologia , Camundongos , NF-kappa B/imunologia , Transdução de Sinais/imunologia
4.
J Cell Biol ; 178(7): 1101-8, 2007 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-17893239

RESUMO

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of "open" chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Dano ao DNA , Animais , Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Mutagênicos/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacos , Telômero/metabolismo
5.
Sci Rep ; 7: 44662, 2017 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-28317934

RESUMO

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Feminino , Células HeLa , Histonas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Masculino , Camundongos Knockout , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/metabolismo , Radiação Ionizante , Proteína de Replicação A/metabolismo
6.
Nat Cell Biol ; 18(12): 1357-1366, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27820601

RESUMO

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.


Assuntos
Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Heterocromatina/metabolismo , Recombinação Homóloga/genética , Humanos , Meiose , Camundongos , Ligação Proteica , Transdução de Sinais , Xenopus
7.
Nat Struct Mol Biol ; 18(6): 721-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21552262

RESUMO

Oncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress. The primary responder to replicative stress is not Ataxia-Telangiectasia Mutated (ATM) but rather the kinase ATM and Rad3-related (ATR). One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates replicative stress, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53-deficient cells, this toxicity being exacerbated by replicative stress-generating conditions such as the overexpression of cyclin E. Notably, one of the compounds we identified is NVP-BEZ235, a dual phosphatidylinositol-3-OH kinase (PI3K) and mTOR inhibitor that is being tested for cancer chemotherapy but that we now show is also very potent against ATM, ATR and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/isolamento & purificação , Proteínas Mutadas de Ataxia Telangiectasia , Sobrevivência Celular , Células Cultivadas , Quebra Cromossômica/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Imidazóis/isolamento & purificação , Imidazóis/farmacologia , Camundongos , Oxazinas/isolamento & purificação , Oxazinas/farmacologia , Quinolinas/isolamento & purificação , Quinolinas/farmacologia , Proteína Supressora de Tumor p53/deficiência
8.
Nat Struct Mol Biol ; 18(12): 1331-1335, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22120667

RESUMO

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia , Estresse Fisiológico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia , Quinase 1 do Ponto de Checagem , Dano ao DNA , Linfoma/tratamento farmacológico , Linfoma/genética , Linfoma/patologia , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
9.
PLoS One ; 4(5): e5475, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19421407

RESUMO

Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Senescência Celular , Proteínas de Ligação a DNA/fisiologia , Fibrossarcoma/prevenção & controle , Genes ras/fisiologia , Neoplasias Pulmonares/prevenção & controle , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adenoma/metabolismo , Adenoma/patologia , Adenoma/prevenção & controle , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metilcolantreno/toxicidade , Camundongos , Camundongos Knockout , Fosforilação , Células Tumorais Cultivadas
10.
Nat Genet ; 41(8): 891-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19620979

RESUMO

Although DNA damage is considered a driving force for aging, the nature of the damage that arises endogenously remains unclear. Replicative stress, a source of endogenous DNA damage, is prevented primarily by the ATR kinase. We have developed a mouse model of Seckel syndrome characterized by a severe deficiency in ATR. Seckel mice show high levels of replicative stress during embryogenesis, when proliferation is widespread, but this is reduced to marginal amounts in postnatal life. In spite of this decrease, adult Seckel mice show accelerated aging, which is further aggravated in the absence of p53. Together, these results support a model whereby replicative stress, particularly in utero, contributes to the onset of aging in postnatal life, and this is balanced by the replicative stress-limiting role of the checkpoint proteins ATR and p53.


Assuntos
Anormalidades Múltiplas/patologia , Envelhecimento/genética , Envelhecimento/patologia , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Progéria/embriologia , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Envelhecimento/efeitos dos fármacos , Alelos , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Encéfalo/enzimologia , Encéfalo/patologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fenótipo , Progéria/enzimologia , Progéria/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Estresse Fisiológico/efeitos dos fármacos , Síndrome , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo
11.
Genes Dev ; 22(3): 297-302, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18245444

RESUMO

The ATR kinase is a key transducer of "replicative stress," the type of genomic damage that has been postulated to be induced by oncogenes. Here we describe a cellular system in which we can unleash ATR activity at will, in the absence of any actual damage or additional signaling pathways triggered by DNA breaks. We demonstrate that activating ATR is sufficient to promote cell cycle arrest and, if persistent, triggers p53-dependent but Ink4a/ARF-independent senescence. Moreover, we show that an ectopic activation of ATR leads to a G1/S arrest in ATM-/- cells, providing the first evidence of functional complementation of ATM deficiency by ATR. Our system provides a novel platform for the study of the specific functions of ATR signaling and adds evidence for the tumor-suppressive potential of the DNA damage response.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Senescência Celular/fisiologia , Quebras de DNA , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/metabolismo
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