Timing of entry of meiosis depends on a mark generated by DNA methyltransferase 3a in testis

Reprogramming of DNA methylation is an essential part of gametogenesis, and a role of two members of the DNA methyltransferase (Dnmt) family, Dnmt3a and Dnmt3L, has been recognized. In an attempt to elucidate the role of Dnmt3a, we analyzed the progression of spermatogenesis in Dnmt3a (-/-) homozygotes during the first 3 weeks of post-natal development. The emerging picture was markedly different from that recently reported for the Dnmt3L protein. In the Dnmt3a (-/-) testis, at the expected time of entry into meiosis (11-13 dpp), the number of spermatocytes was greatly reduced. They progressively accumulated during the following days, but at a slower rate than in the wild type. Once started, however, the pachytene stage was apparently completed with normal chromosome pairing and formation of the sex vesicle, and spermatogenesis further progressed with the appearance and the expression of round spermatid specific markers. Interestingly and unlike Dnmt3L (-/-) spermatocytes, Dnmt3a (-/-) germ cells showed only a minor reduction in the methylation of interspersed repetitive elements and retroposons. The Dnmt3a might thus generate a mark important for the initiation of male meiosis that is distinct from that created by Dnmt3L.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.     

5-Aza-dC在mES细胞向生殖细胞分化中的DNA甲基化调控机制

目的: 探讨小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)向生殖细胞(Embryonic germ cells,EG)分化过程中5-杂氮-2'-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC) 对DNA甲基化转移酶Dnmt1和Dnmt3a及生殖细胞特征基因Mvh表达变化的DNA甲基化调控机制。方法:将mES细胞分化形成拟胚体(embryoid bodies, EBs) 作为向生殖细胞分化的启动步骤,采用不同浓度(0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,3μmol/L)处理EBs,RT-PCR实时荧光定量RT-PCR和Western blot分别检测检测在5-Aza-dC处理前后Dnmt1和Dnmt3a在ES细胞和EBs中的表达,甲基化特异性PCR(MSP)检测原始生殖细胞分化特征基因Mvh启动子甲基化状态。结果: 5-Aza-dC的浓度在0.05 μmol/L~1 μmol/L之间时,EBs保持较高的存活率而EBs的形态明显发生了变化;5-Aza-dC 处理后, Dnmt1和Dnmt3a在EBs中mRNA表达量明显降低,其变化特点与WB结果相一致。MSP和测序结果显示, Mvh启动子区表现为部分甲基化,5-Aza-dC 处理后的4d EBs中Mvh CpG岛有4个CG位点发生突变,而mES细胞中未见突变。结论: EBs经5-Aza-dC处理后,Dnmt1和Dnmt3a的表达明显下调;同时,Mvh启动子发生部分甲基化,有可能启动了向生殖细胞的分化进程。     

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.     

DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages

DNA methylation is necessary for the silencing of endogenous retrotransposons and the maintenance of monoallelic gene expression at imprinted loci and on the X chromosome. Dynamic changes in DNA methylation occur during the initial stages of primordial germ cell development; however, all consequences of this epigenetic reprogramming are not understood. DNA demethylation in postmigratory primordial germ cells coincides with erasure of genomic imprints and reactivation of the inactive X chromosome, as well as ongoing germ cell differentiation events. To investigate a possible role for DNA methylation changes in germ cell differentiation, we have studied several marker genes that initiate expression at this time. Here, we show that the postmigratory germ cell-specific genes Mvh, Dazl and Scp3 are demethylated in germ cells, but not in somatic cells. Premature loss of genomic methylation in Dnmt1 mutant embryos leads to early expression of these genes as well as GCNA1, a widely used germ cell marker. In addition, GCNA1 is ectopically expressed by somatic cells in Dnmt1 mutants. These results provide in vivo evidence that postmigratory germ cell-specific genes are silenced by DNA methylation in both premigratory germ cells and somatic cells. This is the first example of ectopic gene activation in Dnmt1 mutant mice and suggests that dynamic changes in DNA methylation regulate tissue-specific gene expression of a set of primordial germ cell-specific genes.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

Genomic imprinting and epigenetic reprogramming: unearthing the garden of forking paths

Genomic imprinting, an epigenetic form of gene regulation, determines the parent-dependent gene expression of marked or imprinted genes during gametogenesis and embryonic development. Imprinting involves differential allele DNA methylation in one sex cell lineage but not in the other. Egg and sperm each contributes the same DNA sequences to the zygote but epigenetic imprinting of a subset of genes determines that only one of the parent alleles are expressed relative to the parental origin. Primordial germ cells inherit biallelically imprinted genes from maternal and paternal origin and erase their imprints to start de novo monoallelic imprinting during gametogenesis. Epigenetic paternalization is an ongoing process in the mitotically-dividing spermatogonial stem cell and derived meiotically-dividing spermatocyte progeny to endow sperm with imprinted alleles. Epigenetic maternalization is restricted to the oocyte growth phase of folliculogenesis and is unrelated to DNA replication since it takes place while the oocyte remains in the diplotene stage of meiotic prophase I. Sperm and oocyte genomic methylation patterns depend on the activity of DNA methyltransferases (Dnmt). A variant of Dnmt1, designated Dnmt1o, accumulates in oocyte nuclei during the follicular growth phase. Dnmt3L, an isoform of Dnmt3a and Dnmt3b, but lacking enzymatic activity, interacts with Dnmt2a and Dnmt3b and is required for spermatogenesis. In the mouse early zygote, the male pronucleus is demethylated within 4 h of fertilization. Global demethylation takes place gradually up to the morula stage. In the blastocyst, de novo methylation is reestablished in the inner cell mass but not in the trophectoderm. Both the significance of genomic imprinting and the severe developmental defects caused by disrupted Dnmt activity, point to a need for a better understanding of the causes of low cloning efficiency by somatic nuclear transfer to enucleated ovulated oocyte.     

Stat4 limits DNA methyltransferase recruitment and DNA methylation of the IL-18Ralpha gene during Th1 differentiation

Stat4 is required for Th1 development, although how a transiently activated factor generates heritable patterns of gene expression is still unclear. We examined the regulation of IL-18Ralpha expression to define a mechanism for Stat4-dependent genetic programming of a Th1-associated gene. Although Stat4 binds the Il18r1 promoter following IL-12 stimulation and transiently increases acetylated histones H3 and H4, patterns of histone acetylation alone in Th1 cells may not be sufficient to explain cell-type-specific patterns of gene expression. The level of DNA methylation and recruitment of Dnmt3a to Il18r1 inversely correlate with IL-18Ralpha expression, and blocking DNA methylation increases IL-18Ralpha expression. Moreover, there was decreased Il18r1-Dnmt3a association and DNA methylation following transient trichostatin A-induced histone hyperacetylation in Stat4-/-Th1 cultures. Increased association of Dnmt3a and the Dnmt3a cofactor Dnmt3L with the promoters of several Stat4-dependent genes was found in Stat4-/- Th1 cultures, providing a general mechanism for Stat4-dependent gene programming. These data support a mechanism wherein the transient hyperacetylation induced by Stat4 prevents the recruitment of DNA methyltransferases and the subsequent repression of the Il18r1 locus.     

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ~8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length.     

Stimulation effect of Dnmt3L on the DNA methylation activity of Dnmt3a2

Quantification of DNA methyltransferases Dnmt3a and Dnmt3a2, and Dnmt3L in isolated male gonocytes in day 16.5 embryos confirmed that not Dnmt3a but Dnmt3a2 and Dnmt3L were the major Dnmt3s. The expression level of Dnmt3L constituted 5- to 10-fold molar excess compared to that of Dnmt3a2. The stimulation property of the DNA methylation activity of Dnmt3a2 with Dnmt3L towards substrate DNA in naked or nucleosomes was similar to that of Dnmt3a. However, the DNA methylation activity of not Dnmt3a but Dnmt3a2 was severely inhibited at the physiological salt concentration. Interestingly, the activity of Dnmt3a2 was significantly detected in the presence of Dnmt3L even at the physiological salt concentration. This indicates that Dnmt3a2 functions only in the presence of Dnmt3L in male gonocytes, and may explain why Dnmt3L is required specifically in mouse gonocytes for DNA methylation.