RESUMO
Organ size is precisely regulated during development, but the control mechanisms remain obscure. We have isolated a mutation in zebrafish, liebeskummer (lik), which causes development of hyperplastic embryonic hearts. lik encodes Reptin, a component of a DNA-stimulated ATPase complex. The mutation activates ATPase activity of Reptin complexes and causes a cell-autonomous proliferation of cardiomyocytes to begin well after progenitors have fashioned the primitive heart tube. With regard to heart growth, beta-catenin and Pontin, a DNA-stimulated ATPase that is often part of complexes with Reptin, are in the same genetic pathways. Pontin reduction phenocopies the cardiac hyperplasia of the lik mutation. Thus, the Reptin/Pontin ratio serves to regulate heart growth during development, at least in part via the beta-catenin pathway.
Assuntos
Coração/embriologia , Proteínas Nucleares/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Proteases Dependentes de ATP , Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cardiomegalia/embriologia , Cardiomegalia/etiologia , Cardiomegalia/genética , Proteínas de Transporte , Divisão Celular , Sequência Conservada , Proteínas do Citoesqueleto/fisiologia , Embrião não Mamífero , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/fisiologia , Dados de Sequência Molecular , Miocárdio/citologia , Miocárdio/ultraestrutura , Proteínas Nucleares/genética , Mutação Puntual , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/fisiologia , Transativadores/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , beta CateninaRESUMO
DNA methyltransferases, Dnmt3a and Dnmt3b, are required for de novo methylation in embryonic stem (ES) cells and postimplantation embryos. However, the mechanism of de novo methylation is largely unknown. In this study, we have analyzed the sequence specificity of Dnmt3a and Dnmt3b during de novo methylation of murine Moloney leukemia virus provirus DNA in virus-infected ES cells. Provirus DNA from infected wild-type (J1), Dnmt1-/- (c/c), and Dnmt3a3b-/- (3a3b-/-) ES cells were analyzed using the bisulfite sequencing method. We demonstrate that Dnmt3 enzymes methylate predominantly CpG sites in vivo and confirm that Dnmt3 enzymes, but not Dnmt1, are responsible for de novo methylation. However, the sequence context and CpG density do not appear to influence de novo methylation, though strand bias is detectable. Interestingly, non-CpG methylation is detected as a component of de novo methylation. CpA methylation was detected at approximately 1.4% of all sites in J1 and approximately 1.0% in c/c, but only approximately 0.2% in 3a3b-/-. Few methylated CpT or CpC sites were detected. Similar results from nearest neighbor analysis of global endogenous methylation levels indicated a correlation between Dnmt3a and Dnmt3b presence and CpA methylation. These results demonstrate that the Dnmt3 enzymes methylate predominantly CpG sites and at a low frequency CpA sites with no apparent sequence preferences.