TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.

Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.

Tetrachloro-1,4-benzoquinone induces apoptosis of mouse embryonic stem cells

Pentachlorophenol（PCP） is a widespread,persistent environmental contaminant,and it is enzymatically activated to form a reactive metabolite,tetrachloro-l,4-benzoquinone（TCBQ）.To our knowledge,there is no information about TCBQ toxicity on embryonic stem cells.Here,we demonstrated that TCBQ induced significantly apoptosis of mouse embryonic stem cells in a concentration-dependent manner.We also showed that TCBQ elevated genomic5-hydroxymethylcytosine（5hmC） by affecting ten-eleven translocation（Tet） dioxygenases in mouse embryonic stem cells.We further investigated whether Tet dioxygenases were implicated in TCBQ-induced apoptosis.By depleting all three dioxygenases（Tet1-3）,we found that Tet dioxygenases slightly inhibited both early and late apoptosis induced by TCBQ at a low concentration（30 μmol/L）.Meanwhile,treated by TCBQ at higher concentrations（40and 50 μmol/L）,the total percentage of apoptotic cells was not affected by Tet dioxygenases.However,Tet dioxygenases tended to arrest mouse ES cells to be at early apoptotic stage and to reduce the cells to enter later apoptotic stage.These results indicate that Tet dioxygenases play a role in shaping TCBQ-induced apoptosis in mouse embryonic stem cells.Our study provides new insights into the toxicology of PCP and its reactive metabolite TCBQ.

动植物DNA主动去甲基化途径及其调控机制研究进展

甲基化是DNA的一种化学修饰,能够在不改变DNA序列的前提下改变遗传表现,是一种相对稳定且可遗传的表观遗传标记。在生物体内DNA甲基化和去甲基化的动态平衡控制着基因表达的强度。植物和动物细胞中均存在DNA主动去甲基化现象,植物中ROS1/DME切除甲基化的胞嘧啶后由碱基切除修复机制完成DNA主动去甲基化;动物中则是TET1蛋白先将5-甲基胞嘧啶(5-methylcytosine,5mC)氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC)、5-甲酰基胞嘧啶(5-formylcytosine,5fC)和5-羧基胞嘧啶(5-carboxycytosine,5caC),然后通过胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)切除5fC和5caC,最后经过碱基切除修复得到未修饰的胞嘧啶。本文主要对动植物中DNA主动去甲基化途径及其调控机制的最新研究进展进行综述及比较分析,为相关领域的进一步深入研究提供理论支持。

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread,chronic abdominal pain associated with altered bowel movements.Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases.In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1(GATA1)in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation(NCI).The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay,chromatin immunoprecipitation,patch clamp,and interference in vitro and in vivo.In addition,a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island.We showed that NCI caused the induction of GATA1,Ten-eleven translocation 3(TET3),and purinergic receptors(P2X7Rs)in astrocytes of the spinal dorsal horn,and demonstrated that inhibiting these molecules markedly increased the pain threshold,inhibited the activation of astrocytes,and decreased the spinal sEPSC frequency.NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1–TET3 physical interaction and GATA1 binding at the p2x7r promoter.Importantly,we showed that demethylation of the p2x7r locus(and the attendant increase in P2X7R expression)was reversed upon knockdown of GATA1 or TET3 expression,and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter.These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes,and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.