RESUMO
DNA methylation is an important epigenetic modification regulating various biological phenomena, including genomic imprinting and transposon silencing. It is known that methylation of the differentially methylated regions (DMRs) associated with paternally imprinted genes and of some repetitive elements occurs during male germ cell development in the mouse. We have performed a detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2 and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and Line1] and satellite repeats (major and minor) to determine the timing of this de novo methylation in male germ cells. Furthermore, we have examined the roles of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein (Dnmt3L) in this process. We found that methylation of all DMRs and repeats occurred progressively in fetal prospermatogonia and was completed by the newborn stage. Analysis of newborn prospermatogonia from germline-specific Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19 and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed repeats IAP and Line1. Only Dnmt3b was required for the methylation of the satellite repeats. These results indicate both common and differential target specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed moderate to severe hypomethylation in Dnmt3L-deficient prospermatogonia, indicating the critical function and broad specificity of this factor in de novo methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células Germinativas/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Animais , Animais Recém-Nascidos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , DNA Satélite/genética , DNA Satélite/metabolismo , Feminino , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sequências Repetitivas Dispersas/genética , Masculino , Camundongos , Espermatogênese/genética , DNA Metiltransferase 3BRESUMO
BACKGROUND: 1,25-Dihydroxy-22-oxavitamin D(3) (22-oxacalcitriol, OCT) is an analogue of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3), calcitriol) with less calcaemic activity, thus more suitable than 1,25(OH)(2)D(3) for the control of parathyroid hormone (PTH) secretion in chronic dialysis patients. As the low-calcaemic action of OCT has been mainly attributed to its short half-life in the blood stream, the number of doses per week is the key factor to effective OCT therapy toward suppression of PTH secretion and hypercalcaemia. Thus, we investigated a comparison between daily and thrice-weekly i.v. administration of OCT regarding suppression of PTH secretion and calcaemic action in 5/6 nephrectomized rats as a model for chronic renal failure. METHODS: Model rats of chronic renal failure were made by 5/6 nephrectomy. At 3 months after surgery, they were administered either vehicle or OCT intravenously, daily (0.125 or 0.625 microg/kg) or thrice-weekly (0.6 or 3.0 microg/kg) for 2 weeks. RESULTS: The data show that 0.625 microg/kg/day (=4.375 microg/kg/week) suppresses PTH secretion with significant increase in calcium levels at 24 h after the final administration, on the other hand, 3.0 microg/kg/ thrice-weekly (=9.0 microg/kg/week) suppresses PTH secretion, although moderate compared with 0.625 microg/kg/day, with a slight (not significant) increase in calcium. CONCLUSIONS: The current clinical mode of OCT therapy, i.v. thrice-weekly administration, is a practically recommendable protocol.
