Biomedical subjects

Kenichiro Hata

Publications and source records attributed to Kenichiro Hata.

6 recordsLinked to original sources

PubMed2006

Mutations in DNA methyltransferase DNMT3B in ICF syndrome affect its regulation by DNMT3L.

Deficiency in DNA methyltransferase DNMT3B causes a recessive human disorder characterized by immunodeficiency, centromeric instability and facial anomalies (ICF) in association with defects in genomic methylation. The majority of ICF mutations are single amino acid substitutions in the conserved catalytic domain of DNMT3B, which are believed to impair its enzymatic activity directly. The establishment of intact genomic methylation patterns in development requires a fine regulation of the de novo methylation activity of the two related methyltransferases DNMT3A and DNMT3B by regulatory factors including DNMT3L which has a stimulatory effect. Here, we show that two DNMT3B mutant proteins with ICF-causing substitution (A766P and R840Q) displayed a methylation activity similar to the wild-type enzyme both in vitro and in vivo. However, their stimulation by DNMT3L was severely compromised due to deficient protein interaction. Our findings suggest that methylation defects in ICF syndrome may also result from impaired stimulation of DNMT3B activity by DNMT3L or other unknown regulatory factors as well as from a weakened basal catalytic activity of the mutant DNMT3B protein per se.

Amino Acid Substitution↗

PubMed2006

Stochastic imprinting in the progeny of Dnmt3L-/- females.

The cis-acting regulatory sequences of imprinted genes are subject to germline-specific epigenetic modifications, the imprints, so that this class of genes is exclusively expressed from either the paternal or maternal allele in offspring. How genes are differentially marked in the germlines remains largely to be elucidated. Although the exact nature of the mark is not fully known, DNA methylation [at differentially methylated regions (DMRs)] appears to be a major, functional component. Recent data in mice indicate that Dnmt3a, an enzyme with de novo DNA methyltransferase activity, and the related protein Dnmt3L are required for methylation of imprinted loci in germ cells. Maternal methylation imprints, in particular, are strictly dependent on the presence of Dnmt3L. Here, we show that, unexpectedly, methylation imprints can be present in some progeny of Dnmt3L(-/-) females. This incomplete penetrance of the effect of Dnmt3L deficiency in oocytes is neither embryo nor locus specific, but stochastic. We establish that, when it occurs, methylation is present in both embryo and extra-embryonic tissues and results in a functional imprint. This suggests that this maternal methylation is inherited, directly or indirectly, from the gamete. Our results indicate that in the absence of Dnmt3L, factors such as Dnmt3a and possibly others can act alone to mark individual DMRs. However, establishment of appropriate maternal imprints at all loci does require a combination of all factors. This observation can provide a basis to understand mechanisms involved in some sporadic cases of imprinting-related diseases and polymorphic imprinting in human.

Animals↗

PubMed2006

Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells.

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002). Also, a paternally imprinted gene, H19, loses DNA methylation in Dnmt3L-/- spermatogonia (Bourc'his and Bestor, 2004; Kaneda et al., 2004). To determine the reason for the impaired spermatogenesis in the Dnmt3L-/- testes, we have carried out a series of histological and molecular studies. We show here that Dnmt3L-/- germ cells were arrested and died around the early meiotic stage. A microarray-based gene expression-profiling analysis revealed that various gonad-specific and/or sex-chromosome-linked genes were downregulated in the Dnmt3L-/- testes. In contrast, expression of retrovirus-like intracisternal A-particle (IAP) sequences was upregulated; consistent with this observation, a specific IAP copy showed complete loss of DNA methylation. These findings indicate that Dnmt3L regulates germ cell-specific gene expression and IAP suppression, which are critical for male germ cell proliferation and meiosis.

Animals↗

PubMed2004

Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting.

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.

Alleles↗

PubMed2002

Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.

Genomic imprinting is regulated by differential methylation of the paternal and maternal genome. However, it remains unknown how parental imprinting is established during gametogenesis. In this study, we demonstrate that Dnmt3L, a protein sharing homology with DNA methyltransferases, Dnmt3a and Dnmt3b, but lacking enzymatic activity, is essential for the establishment of maternal methylation imprints and appropriate expression of maternally imprinted genes. We also show that Dnmt3L interacts with Dnmt3a and Dnmt3b and co-localizes with these enzymes in the nuclei of transfected cells, suggesting that Dnmt3L may regulate genomic imprinting via the Dnmt3 family enzymes. Consistent with this model, we show that [Dnmt3a(-/-), Dnmt3b(+/-)] mice also fail to establish maternal methylation imprints. In addition, both Dnmt3a and Dnmt3L are required for spermatogenesis. Together, our findings suggest that Dnmt3L may cooperate with Dnmt3 family methyltransferases to carry out de novo methylation of maternally imprinted genes in oocytes.

3T3 Cells↗

PubMed2002

[Injectable bone].

Tissue engineering applies the principles of biology and engineering to the development of functional substitutes,for example 1) cells, 2) scaffolds, 3) some cytokines, for lost tissues. The meaning of this bone regeneration is that it decreases the needed tissues and burden of patients. In this time, we applied to mesenchymal stem cells (MSCs) from the own bone marrow as cell sources. MSCs are thought to be multipotent cells that can replicate. And we also used a beta-tricalcium phosphate (beta-TCP) as a scaffold and fibrin glue as materials to regenerate a injectable bone and we injected into the subcutaneous space on the dorsum of the rat. After 8 weeks of implantation, it could be confirmed newly formed bone and fibrin glue functioned as a injectable materials without loosing the cell activity and the proliferation of MSCs. Next we applied Platelet Rich Plasma (PRP) to improve the ability of osteogenesis. PRP contains some cytokines and are expected to promote the increase of osteogenesis. The merit is not immunity rejection from autologous blood collected in the immediate preoperative period. The admixtures of PRP or fibrin glue have fluidity and gel-like consistency as the thrombin mixing. They can be injected with a syringe in tissues. We named this "Injectable Bone". According to the histological observations, the MSCs with PRP were well formed mature bone and neovascularization compared with control (defect only) after 8 weeks implantation. These results demonstrated that the mixture of MSCs and PRP were useful as injectable bone substitute and its ability of bone regeneration is almost equal to autogenous particulate cancellous bone and marrow (PCBM).

English Abstract↗