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 remodel-ing 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 establish-ment 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 postim-plantation 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.

Influence of DNA methyltransferase 3b on FHIT expression and DNA methylation of the FHIT promoter region in hepatoma SMMC-7721 cells

BACKGROUND: Alterations in DNA methylation occur during the pathogenesis of human tumors. In this study, we investigated the influence of DNA methyltransferase 3b (DNMT3b) on fragile histidine trial (FHIT) expression and on DNA methylation of the FHIT promoter region in the hepatoma cell line SMMC-7721. METHODS: DNMT3b siRNA was used to down-regulate DNMT3b expression. DNMT3b and FHIT proteins were determined by Western blotting. Methylation-specific PCR was used to analyze the methylation status of the FHIT gene. RESULTS: After DNMT3b siRNA transfection, the expression of DNMT3b was inhibited in SMMC-7721 cells, and the expression of FHIT was significantly higher than that in the control group. There was no significant difference in methylation status between the DNMT3b siRNA transfected cells and control cells. CONCLUSION: DNMT3b may play an important role in regulation of FHIT expression in hepatoma SMMC-7721 cells, but not through methylation of the FHIT promoter. (Hepatobiliary Pancreat Dis Int 2009; 8: 273-277)

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees(Apis mellifera)

In honeybee （Apis mellifera） colonies, queens and workers are altemative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR （target of rapamycin） and Dnmt3 （DNA methyltransferase 3） genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K （similar to RPS6-p70-protein kinase） gene linked closely to organismal growth and development in queen and worker larvae （1-day and 3-day old）. Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

DNA甲基转移酶DNMT3A在人类常见病毒感染中的研究进展

DNA甲基转移酶(DNA methylationtransferases,DNMTs)是哺乳动物建立与维持基因甲基化的酶类家族,参与基因表达和调控等生物学过程。其中DNA甲基转移酶3A(DNA methyltransferase 3 Alpha,DNMT3A)是机体重要DNMTs之一,DNMT3A突变或异常表达所诱导的基因甲基化引起机体相关因子活性失调进而诱发疾病发生,DNMT3A介导的基因甲基化与人类常见病毒感染所致疾病密切相关。本篇综述从人类常见病毒感染宿主的角度出发,对DNMT3A在促进病毒感染与诱发疾病中的作用进行阐述,为进一步探究以DNMT3A为病毒感染性疾病治疗靶点提供参考和思路。

Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract carcinoma cells

BACKGROUND: Hypermethylation of the promoter region is one of the major mechanisms of tumor suppressor gene inactivation. DNA methyltransferase 3b (DNMT3b), an enzyme that participates in the establishment of de novo methylation patterns, is highly expressed in many tumor cells and tissues, and it is closely associated with hypermethylation of the promoter of tumor suppressor genes. The aim of this study was to explore the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the expression of the DNMT3b gene in human biliary tract carcinoma cell. METHODS: The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into the human biliary tract carcinoma cell line QBC-939 with lipofectamine transfection reagent, and positive cell clones were formed using G418 selection after transfection. The constructed recombinant plasmid was transfected into QBC-939 cells successfully and was confirmed by amplification of the exogenous neo^R gene with the polymerase chain reaction method. The expression of DNMT3b gene mRNA and protein was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry respectively. RESULTS: Following transfection, the mRNA level of the DNMT3b gene decreased from 0.956±0.053 to 0.209±0.023, and the protein level of the DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. Very significant differences were observed both at the transcription and posttranscription levels in the expression of the DNMT3b gene between the non-tranfection group and the antisense DN- MT3b gene eukaryotic expression plasmid transfection group (P<0.01). CONCLUSIONS: Transfection with the antisense DNMT3b gene eukaryotic expression plasmid can significantly reduce the expression level of the DNMT3b gene in the human biliary tract carcinoma cell line QBC-939. This study may provide a valid method to investigate the function of the DNMT3b gene and its role in biliary tract carcinoma.

Effects of Bufalin on Up-regulating Methylation of Wilm's Tumor 1 Gene in Human Erythroid Leukemic Cells

Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. Results The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC50) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Conclusions Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G0/G1 phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.