Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

Dynamic methylation pattern of the methyltransferase1o (Dnmt1o) 5′‐flanking region during mouse oogenesis and spermatogenesis

DNA methyltransferase1o (Dnmt1o), which is specific to oocyte and preimplantation embryo, plays a role in maintaining DNA methylation in mammalian cells. Here, we investigated the methylation status of CpGs sites in the Dnmt1o 5′‐flanking region in germ cells at different stages of oogenesis or spermatogenesis. The methylation levels of the CpG sites at the 5′‐flanking regions were hypermethylated in growing oocytes of all follicular stages, while the oocytes in meiotic metaphase II (MII) were demethylated. The methylation pattern within the CpGs sites in the 5′‐flanking region, however, was dramatically changed during spermatogenesis. We observed that there was significant non‐CpG methylation both in MII oocytes and spermatocytes. Although a low methylation level in non‐CpG sites was observed in primary and secondary oocytes, the CpA site of position 25 and CpT site of position 29 within the no‐CpG region in the 5′‐flanking region of Dnmt1o was highly methylated in MII oocytes. During spermatogenesis, the low degree of methylation at CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of methylation in non‐CpG sites in spermatocytes decreased. Together, germ cells showed inverted methylation patterns between CpG and non‐CpG sites in the Dnmt1o 5′‐upstream region, and the methylation pattern during oogenesis did not drastically change, remaining generally hypomethylated at the MII stage. Mol. Reprod. Dev. 80: 212–222, 2013. © 2013 Wiley Periodicals, Inc.     

Aberrant DNA methylation imprints in aborted bovine clones

Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.     

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.     

哺乳动物DNA甲基化及其在体细胞诱导重编程中的作用

诱导多能干细胞(Induced pluripotent stem cell, iPS)技术提供了将终末分化的细胞逆转为多潜能干细胞的可能, 在干细胞基础理论研究和再生医学中具有重要意义。然而, 目前体细胞诱导重编程方法效率极低, 常发生不完全的重编程。研究表明, 在不完全重编程的细胞中存在体细胞的表观遗传记忆, 而DNA甲基化作为相对长期和稳定的表观遗传修饰, 是影响重编程效率和iPS细胞分化能力的重要因素之一。哺乳动物DNA甲基化是指胞嘧啶第五位碳原子上的甲基化修饰, 常发生于CpG位点。DNA甲基化能够调节体细胞特异基因和多能性基因的表达, 因此其在哺乳动物基因调控、胚胎发育和细胞重编程过程中发挥着重要作用。此外, 异常DNA甲基化可能导致iPS细胞基因印记的异常和X染色体的失活。文章重点围绕DNA甲基化的机制、分布特点、及其在体细胞诱导重编程中的作用进行了综述。     

Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells

To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.     

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.     

Cloning and sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-methyltransferase-1

The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms.     

Knockdown of sensitive to apoptosis gene by small interfering RNA enhances the sensitivity of PC3 cells toward actinomycin D and etoposide

Abstract

Actinomycin D and etoposide induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Sensitive to apoptosis gene (SAG) protein, a redox inducible zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger. The present report show that knockdown of SAG expression in PC3 cells greatly enhances apoptosis induced by actinomycin D and etoposide. Transfection of human prostate cancer PC3 cells with SAG small interfering RNA (siRNA) markedly decreased the expression of SAG, enhancing the susceptibility of actinomycin D- and etoposide-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that SAG may play an important role in regulating the apoptosis induced by actinomycin D and etoposide and the sensitizing effect of SAG siRNA on the apoptotic cell death of PC3 cells offers the possibility of developing a modifier of cancer chemotherapy.     

DNA甲基化和组蛋白修饰在克隆动物发育过程中的作用

体细胞核移植在农业应用、生产疾病模型动物、转基因家畜或产生人胚胎干细胞来治疗人类的疾病方面有巨大的应用潜力。虽然已经成功克隆出多种哺乳动物, 但该技术仍存在一些未解决的问题, 包括产生克隆动物的效率低和克隆动物的异常等。异常的表观遗传重编程是克隆胚胎发育失败的一个重要因素。文章重点论述了DNA甲基化、组蛋白修饰及其与克隆胚胎发育的关系。了解表观遗传调控机制有助于解决核移植技术中存在的问题, 有利于更好地应用这项技术。