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1.
Cell ; 158(2): 288-299, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25036629

RESUMO

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Carboidratos da Dieta/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Butiratos/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Pólipos do Colo/metabolismo , Pólipos do Colo/microbiologia , Pólipos do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Reparo de Erro de Pareamento de DNA , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/metabolismo , Organismos Livres de Patógenos Específicos , beta Catenina/metabolismo
2.
Genes Dev ; 34(11-12): 806-818, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32354835

RESUMO

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5' → 3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.


Assuntos
Reparo do DNA/fisiologia , Meiose , Animais , Cromatina/química , Cromatina/metabolismo , DNA/química , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Estrutura Molecular , Recombinação Genética
3.
Nature ; 589(7841): 293-298, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33299182

RESUMO

H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Epigênese Genética , Histonas/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Cromatina/química , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Inativação Gênica , Histonas/química , Ativação Linfocitária/genética , Masculino , Metilação , Camundongos , Camundongos Knockout
4.
J Biol Chem ; 300(1): 105492, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38000655

RESUMO

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.


Assuntos
Desoxiadenosinas , Metionina Adenosiltransferase , Neoplasias , Proteína-Arginina N-Metiltransferases , Purina-Núcleosídeo Fosforilase , S-Adenosilmetionina , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxiadenosinas/antagonistas & inibidores , Desoxiadenosinas/genética , Desoxiadenosinas/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Neoplasias/genética , Neoplasias/fisiopatologia , Neoplasias/terapia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/farmacologia , Pirrolidinas/uso terapêutico , S-Adenosilmetionina/metabolismo
5.
Brain ; 147(5): 1784-1798, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38387080

RESUMO

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.


Assuntos
Proteína Huntingtina , Doença de Huntington , Expansão das Repetições de Trinucleotídeos , Doença de Huntington/genética , Doença de Huntington/terapia , Animais , Humanos , Expansão das Repetições de Trinucleotídeos/genética , Camundongos , Proteína Huntingtina/genética , Proteína 3 Homóloga a MutS/genética , Modelos Animais de Doenças , Proteínas do Tecido Nervoso/genética , Encéfalo/patologia , Encéfalo/metabolismo
6.
Nucleic Acids Res ; 50(14): 8093-8106, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35849338

RESUMO

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.


Assuntos
Enzimas Reparadoras do DNA , Reparo do DNA , Exodesoxirribonucleases , Neoplasias , Animais , Linfócitos B , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Imunidade , Meiose/genética , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Hipermutação Somática de Imunoglobulina
7.
Gastroenterology ; 161(4): 1288-1302.e13, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34224739

RESUMO

BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone. CONCLUSIONS: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.


Assuntos
Antígenos de Neoplasias/farmacologia , Vacinas Anticâncer/farmacologia , Neoplasias Colorretais Hereditárias sem Polipose/tratamento farmacológico , Mutação da Fase de Leitura , Fenômenos Imunogenéticos , Fragmentos de Peptídeos/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/imunologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Epitopos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Homóloga a MutS/genética , Naproxeno/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral , Vacinação , Eficácia de Vacinas
8.
Hum Mol Genet ; 28(11): 1755-1767, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615115

RESUMO

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q+/+) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q+/+ mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood-brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.


Assuntos
Hidroximetilbilano Sintase/genética , Doenças do Sistema Nervoso/genética , Porfiria Aguda Intermitente/genética , Transtornos Psicomotores/genética , Ácido Aminolevulínico/sangue , Ácido Aminolevulínico/urina , Animais , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Técnicas de Introdução de Genes , Genes Dominantes , Homozigoto , Humanos , Hidroximetilbilano Sintase/metabolismo , Fígado/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Doenças do Sistema Nervoso/sangue , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/urina , Fenobarbital/farmacologia , Porfobilinogênio/sangue , Porfobilinogênio/urina , Porfiria Aguda Intermitente/sangue , Porfiria Aguda Intermitente/patologia , Porfiria Aguda Intermitente/urina , Transtornos Psicomotores/sangue , Transtornos Psicomotores/patologia , Transtornos Psicomotores/urina
9.
PLoS Genet ; 14(10): e1007719, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30312299

RESUMO

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5' end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSß) and MSH2/MSH6 (MutSα), together with Polß, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSß in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans.


Assuntos
Enzimas Reparadoras do DNA/genética , Exodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Proteínas MutL/genética , Animais , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Reparo do DNA , Enzimas Reparadoras do DNA/fisiologia , Modelos Animais de Doenças , Exodesoxirribonucleases/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Instabilidade Genômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Mutação , Expansão das Repetições de Trinucleotídeos/genética
10.
Mol Cell ; 46(5): 691-704, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22681890

RESUMO

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.


Assuntos
Epistasia Genética , Evolução Molecular , Genes Fúngicos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Fúngico , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Especificidade da Espécie
11.
Development ; 143(11): 1937-47, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27246713

RESUMO

Ocular lens morphogenesis is a model for investigating mechanisms of cellular differentiation, spatial and temporal gene expression control, and chromatin regulation. Brg1 (Smarca4) and Snf2h (Smarca5) are catalytic subunits of distinct ATP-dependent chromatin remodeling complexes implicated in transcriptional regulation. Previous studies have shown that Brg1 regulates both lens fiber cell differentiation and organized degradation of their nuclei (denucleation). Here, we employed a conditional Snf2h(flox) mouse model to probe the cellular and molecular mechanisms of lens formation. Depletion of Snf2h induces premature and expanded differentiation of lens precursor cells forming the lens vesicle, implicating Snf2h as a key regulator of lens vesicle polarity through spatial control of Prox1, Jag1, p27(Kip1) (Cdkn1b) and p57(Kip2) (Cdkn1c) gene expression. The abnormal Snf2h(-/-) fiber cells also retain their nuclei. RNA profiling of Snf2h(-/) (-) and Brg1(-/-) eyes revealed differences in multiple transcripts, including prominent downregulation of those encoding Hsf4 and DNase IIß, which are implicated in the denucleation process. In summary, our data suggest that Snf2h is essential for the establishment of lens vesicle polarity, partitioning of prospective lens epithelial and fiber cell compartments, lens fiber cell differentiation, and lens fiber cell nuclear degradation.


Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Cristalino/citologia , Cristalino/embriologia , Animais , Autofagia , Compartimento Celular , Ciclo Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição de Choque Térmico , Camundongos Knockout , Mitofagia , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genética
12.
Stem Cells ; 35(6): 1614-1623, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28276606

RESUMO

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.


Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Adenosina Trifosfatases/deficiência , Anemia/patologia , Animais , Ciclo Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/deficiência , Dano ao DNA/genética , Células Eritroides/citologia , Eritropoese , Deleção de Genes , Genótipo , Hematopoese , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/metabolismo
13.
Nucleic Acids Res ; 43(15): 7371-87, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26160886

RESUMO

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.


Assuntos
Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA , Desenvolvimento Embrionário , Exodesoxirribonucleases/fisiologia , Proteínas Nucleares/genética , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/toxicidade , Células Cultivadas , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Replicação do DNA , Proteínas de Ligação a DNA , Desenvolvimento Embrionário/genética , Exodesoxirribonucleases/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Deleção de Genes , Genes Letais , Camundongos , Mutação
15.
Semin Immunol ; 24(4): 293-300, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22703640

RESUMO

The creation of a highly diverse antibody repertoire requires the synergistic activity of a DNA mutator, known as activation-induced deaminase (AID), coupled with an error-prone repair process that recognizes the DNA mismatch catalyzed by AID. Instead of facilitating the canonical error-free response, which generally occurs throughout the genome, DNA mismatch repair (MMR) participates in an error-prone repair mode that promotes A:T mutagenesis and double-strand breaks at the immunoglobulin (Ig) genes. As such, MMR is capable of compounding the mutation frequency of AID activity as well as broadening the spectrum of base mutations; thereby increasing the efficiency of antibody maturation. We here review the current understanding of this MMR-mediated process and describe how the MMR signaling cascade downstream of AID diverges in a locus dependent manner and even within the Ig locus itself to differentially promote somatic hypermutation (SHM) and class switch recombination (CSR) in B cells.


Assuntos
Diversidade de Anticorpos , Citidina Desaminase/imunologia , Reparo de Erro de Pareamento de DNA , Animais , Citidina Desaminase/genética , Desaminação , Humanos , Mutação , Ubiquitinação
16.
Proc Natl Acad Sci U S A ; 110(27): E2470-9, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23754438

RESUMO

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Reparo do DNA por Junção de Extremidades/genética , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Exodesoxirribonucleases/deficiência , Exodesoxirribonucleases/genética , Feminino , Masculino , Meiose/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Homologia de Sequência de Aminoácidos
17.
Gut ; 64(12): 1905-12, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25429050

RESUMO

OBJECTIVE: Lynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2(loxP/loxP) Villin-Cre mouse model for Lynch syndrome. DESIGN: Flow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM). RESULTS: Besides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2(loxP/loxP) Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours. CONCLUSIONS: Mesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2(loxP/loxP) Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Benzoquinonas/uso terapêutico , Neoplasias Colorretais Hereditárias sem Polipose/prevenção & controle , Mesalamina/uso terapêutico , Proteína 2 Homóloga a MutS/genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Benzoquinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Modelos Animais de Doenças , Feminino , Mutação da Fase de Leitura , Células HCT116 , Humanos , Mucosa Intestinal/metabolismo , Masculino , Mesalamina/farmacologia , Camundongos , Instabilidade de Microssatélites/efeitos dos fármacos , Proteína 2 Homóloga a MutS/metabolismo , Taxa de Mutação , Carga Tumoral/efeitos dos fármacos
18.
Mol Carcinog ; 54(11): 1376-86, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25213383

RESUMO

Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis.


Assuntos
Pareamento Incorreto de Bases/genética , Reparo de Erro de Pareamento de DNA/genética , Mutação da Fase de Leitura/genética , Neoplasias Intestinais/genética , Repetições de Microssatélites/genética , Animais , Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA , Humanos , Camundongos , Instabilidade de Microssatélites
19.
Dev Biol ; 377(1): 213-23, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23384561

RESUMO

In many mammalian species, more than half of the initial oocyte population is eliminated by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this major oocyte loss remains poorly understood. We examined the apoptotic pathway involved in oocyte elimination in wild-type mouse ovaries as well as in Msh5 -/- ovaries, in which all oocytes were eliminated due to a lack of double strand break repair. Immunoblot and immunofluorescence staining showed that an initiator caspase 9 and an effector caspase 7 were constitutively activated in almost all oocytes in fetal ovaries regardless of their genotypes. In caspase 9 -/- ovaries, the total number of oocytes remained high while that in wild-type ovaries steadily declined during ovarian development. Therefore, the activation of caspase 9 was required for but did not immediately lead to oocyte demise. We found that XIAP, an endogenous inhibitor of apoptosis, was also abundant in oocytes during meiotic prophase progression. On the other hand, a cleaved form of PARP1, a target of effector caspases, was localized to the nuclei of a limited number of oocytes, and the frequency of cleaved PARP1-positive oocyte nuclei increased significantly higher before all oocytes disappeared in Msh5 -/- ovaries. We conclude that the mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated in oocytes and renders the elimination of oocytes with meiotic errors, which can be captured by the cleavage of PARP1.


Assuntos
Caspase 9/metabolismo , Prófase Meiótica I , Oócitos/citologia , Oócitos/enzimologia , Animais , Animais Recém-Nascidos , Caspase 7/metabolismo , Caspase 9/deficiência , Contagem de Células , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Feminino , Feto/citologia , Imunofluorescência , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Ovário/citologia , Ovário/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
20.
Cancer Cell ; 9(6): 417-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16766259

RESUMO

The DNA mismatch repair (MMR) system maintains genome integrity by correcting replication errors. MMR also stimulates checkpoint and cell death responses to DNA damage suggested by the resistance of MMR-defective tumor cells to several chemotherapeutic agents. MMR-dependent cytotoxic response may result from futile repair; however, MMR-mediated apoptosis has been genetically separated from its repair function. In a recent issue of Molecular Cell, Yoshioka and coworkers show that MMR complexes (MutSalpha and MutLalpha) are required for the recruitment of ATR-ATRIP to sites of alkylation damage, demonstrating that MMR complexes can function as sensors in DNA damage signal transduction.


Assuntos
Apoptose , Dano ao DNA , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/fisiologia , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Alquilação , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Pareamento Incorreto de Bases , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA , Humanos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteínas Serina-Treonina Quinases/metabolismo
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