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1.
Hum Mol Genet ; 20(16): 3241-55, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21636528

RESUMO

DNA methyltransferase 1 (DNMT1) maintains methylation at CpG dinucleotides, important for transcriptional silencing at many loci. It is also implicated in stabilizing repeat sequences: DNMT1 deficiency causes microsatellite instability in mouse embryonic stem cells, but it is unclear how this occurs, how repeats lacking CpG become unstable and whether the effect is confined to stem cells. To address these questions, we transfected hTERT-immortalized normal human fibroblasts (hTERT-1604) with a short hairpin RNA construct targeting DNMT1 and isolated stable integrants with different levels of protein. DNMT1 expression levels agreed well with methylation levels at imprinted genes. Knockdown cells showed two key characteristics of mismatch repair (MMR) deficiency, namely resistance to the drug 6-thioguanine and up to 10-fold elevated mutation rates at a CA(17) microsatellite reporter, but had limited viability. The likely cause of MMR defects is a matching drop in steady-state protein levels for key repair components in DNMT1 knockdown cells, affecting both the MutLα and MutSα complexes. This indirect effect on MMR proteins was also seen using a different targeting method in HT29 colon cancer cells and did not involve transcriptional silencing of the respective genes. Decreased levels of MMR components follow activation of the DNA damage response and blocking this response, and in particular poly(ADP-ribose) polymerase (PARP) overactivation, rescues cell viability in DNMT1-depleted cells. These results offer an explanation for how and why unmethylated microsatellite repeats can be destabilized in cells with decreased DNMT1 levels and uncover a novel and important role for PARP in this process.


Assuntos
DNA (Citosina-5-)-Metiltransferases/deficiência , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/metabolismo , Fibroblastos/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Endodesoxirribonucleases/metabolismo , Humanos , Camundongos , Proteína 1 Homóloga a MutL , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
2.
Reprod Biol Endocrinol ; 8: 22, 2010 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-20210997

RESUMO

BACKGROUND: Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. METHODS: To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. RESULTS: FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. CONCLUSIONS: Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.


Assuntos
Imunofilinas/genética , Infertilidade Masculina/genética , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Estudos de Coortes , Ligação Genética , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação/fisiologia , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Tacrolimo , Análise Serial de Tecidos
3.
Genomics ; 84(1): 193-204, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15203217

RESUMO

DNA (cytosine-5-)-methyltransferase genes are important for normal development in mice and humans. We describe here 11 pseudogenes spread among human, mouse, and rat belonging to this gene family, ranging from 1 pseudogene in humans to 7 in rat, all belonging to the Dnmt3 subfamily. All except 1 rat Dnmt3b pseudogene appear to be transcriptionally silent. Dnmt3a2, a transcript variant of Dnmt3a starting at an alternative promoter, had the highest number of processed pseudogenes, while none were found for the canonical Dnmt3a, suggesting the former transcript is more highly expressed in germ cells. Comparison of human, mouse, and rat Dnmt3a2 sequences also suggests that human exon 8 is a recent acquisition. Alignment of the 3'UTR of Dnmt3a2 among the functional genes and the processed pseudogenes suggested that a second polyadenylation site downstream of the RefSeq poly(A) was being used in mice, resulting in a longer 3'UTR, a finding confirmed by RT-PCR in mouse tissues. We also found conserved cytoplasmic polyadenylation elements, usually implicated in regulating translation in oocytes, in Dnmt3b and Dnmt1. Expression of DNMT3B in the mouse oocyte was confirmed by immunocytochemistry. These results clarify the structure of a number of loci in the three species examined and provide some useful insights into the structure and evolution of this gene family.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Família Multigênica/genética , Pseudogenes/genética , Locos de Características Quantitativas/genética , Sinais de Poliadenilação na Ponta 3' do RNA/genética , Transcrição Gênica/genética , Regiões 3' não Traduzidas/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Oócitos/metabolismo , Ratos , DNA Metiltransferase 3B
4.
Mol Cell ; 12(5): 1225-37, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14636580

RESUMO

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fosfoproteínas Fosfatases/metabolismo , Proteína Quinase C/metabolismo , Subunidades Proteicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/fisiologia , Linhagem Celular , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Dimerização , Genes Reporter , Sequências Hélice-Alça-Hélice , Humanos , Dados de Sequência Molecular , Morfogênese/fisiologia , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 2 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Peixe-Zebra
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