Iron deficiency and its epigenetic effects on iron homeostasis

Iron deficiency is a common micronutrient deficiency associated with metabolic changes in the levels of iron regulatory proteins, hepcidin and ferroportin. Studies have associated dysregulation of iron homeostasis to other secondary and life-threatening diseases including anaemia, neurodegeneration and metabolic diseases. Iron deficiency plays a critical role in epigenetic regulation by affecting the Fe²⁺/α-ketoglutarate-dependent demethylating enzymes, Ten Eleven Translocase 1–3 (TET 1–3) and Jumonji-C (JmjC) histone demethylase, which are involved in epigenetic erasure of the methylation marks on both DNA and histone tails, respectively. In this review, studies involving epigenetic effects of iron deficiency associated with dysregulation of TET 1–3 and JmjC histone demethylase enzyme activities on hepcidin/ferroportin axis are discussed.     

TET蛋白的去甲基化机制及其在调控小鼠发育过程中的作用

TET(Ten-eleven translocation)蛋白家族共有3个成员,分别为TET1、TET2和TET3,均属于α-酮戊二酸(α-KG)和Fe²⁺依赖的双加氧酶,可以将5-甲基胞嘧啶(5-methylcytosine, 5 mC)氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5 hmC)、5-甲酰基胞嘧啶(5-formylcytosine, 5 fC)及5-羧基胞嘧啶(5-carboxylcytosine, 5 caC)。研究表明,TET蛋白通过不同机制以主动或被动的方式调控DNA去甲基化,且去甲基化的活性可能受其他因子的调控。TET蛋白广泛参与哺乳动物发育过程的调节,其中在原始生殖细胞的形成、胚胎发育、干细胞多能性及神经和脑发育等方面发挥了重要作用。TET蛋白生物功能的发现为表观遗传学研究开辟了全新的研究领域,而且相关研究结果对拓展生命科学研究具有重要意义。文章综述了TET蛋白家族的结构、去甲基化分子机制及在小鼠发育过程中的作用,为深入了解TET蛋白的功能提供理论基础。     

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.     

TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis

The family of Ten-Eleven Translocation (TET) proteins is implicated in the process of active DNA demethylation and thus in epigenetic regulation. TET 1, 2 and 3 proteins are oxygenases that can hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). The base excision repair (BER) pathway removes the resulting 5-fC and 5-caC bases paired with a guanine and replaces them with regular cytosine. The question arises whether active modification of 5-mC residues and their subsequent elimination could affect the genomic DNA stability. Here, we generated two inducible cell lines (Ba/F3-EPOR, and UT7) overexpressing wild-type or catalytically inactive human TET2 proteins. Wild-type TET2 induction resulted in an increased level of 5-hmC and a cell cycle defect in S phase associated with higher level of phosphorylated P53, chromosomal and centrosomal abnormalities. Furthermore, in a thymine-DNA glycosylase (Tdg) deficient context, the TET2-mediated increase of 5-hmC induces mutagenesis characterized by GC > AT transitions in CpG context suggesting a mutagenic potential of 5-hmC metabolites. Altogether, these data suggest that TET2 activity and the levels of 5-hmC and its derivatives should be tightly controlled to avoid genetic and chromosomal instabilities. Moreover, TET2-mediated active demethylation might be a very dangerous process if used to entirely demethylate the genome and might rather be used only at specific loci.     

TET蛋白去甲基化作用的相关研究进展

TET(ten-eleven translocation)蛋白属于酮戊二酸和Fe2+依赖的双加氧酶,能够产生催化氧化作用。在TET蛋白家族的催化氧化作用下5-甲基胞嘧啶(5-methylcytosine,5mC)可转化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hmC),并可进一步转化为5-甲酰胞嘧啶(5-formylcytosine,5fC)和5-羧基胞嘧啶(5-carboxylcytosine,5caC)。TET蛋白在DNA胞嘧啶的去甲基化、胚胎发育和基因重新编码等过程都存在重要作用,其中TET蛋白参与DNA胞嘧啶的去甲基化过程的作用机制一直是研究热点,另外,有研究发现TET与肿瘤的发生也存在联系,可能成为新的肿瘤分子标志。     

A network-based analysis of the human TET Gene Family

Ten-eleven translocation (TET) proteins, a family of Fe²⁺- and 2-oxoglutarate-dependent dioxygenases, are involved in DNA demethylation. Three TET paralogs have been identified (TET1, TET2, and TET3) and they show different patterns of tissue-specific expression. In our previous evolutionary studies, we found that the TET1 and TET2 genes underwent positive selection more frequently than the TET3 gene, possibly due to changes in the selective constraints during their evolutionary process. In this study, we performed a network-based analysis of the mRNA expression profiles of TET knockdown and the TET-containing co-expression modules identified in early human developmental stages. Analyses based on the PPI subnetwork demonstrated that TET DEGs PPI subnetwork genes were more evolutionarily conserved than all the human-chimpanzee orthologs during evolutionary history. GO annotation of gene co-expression modules containing a TET gene ortholog revealed particular features of the potential role of TET gene family members. Our study implicated the TET1 module in fundamental aspects of cellular physiology, such as the regulation of glucose metabolism, and the TET2 module in GPCR signal transduction. The TET3 module was related to signaling pathways involved in developmental regulation. The evolutionary rate and phylogenetic age distribution analysis of network member genes also support these network-based analyses. The present study provides an integrated view of TET gene family properties and might be informative for elucidating the molecular mechanisms of their biological functions.     

TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells

TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.     

TET家族DNA羟化酶与5-hmC在肿瘤中的作用机制的研究进展

DNA甲基化失调引起基因表达异常是表观遗传学的一个显著特点。目前已知,由DNA甲基转移酶（DNA methyltransferases,DMNTs）催化DNA甲基化,其酶基因突变或表达异常引起DNA甲基化水平的改变。近期研究发现了一种DNA去甲基化酶--TET（Ten-Eleventranslocation）家族DNA羟化酶,能通过多种途径催化5-甲基胞嘧啶（5．methylcytosine,5-mC）去甲基化,从而调控DNA基化的平衡。5-羟甲基胞嘧啶（5-hydroxymethylcytosine,5-hmC）作为DNA去甲基化多重步骤中重要的中间产物,其水平在肿瘤的发生和发展时期发生显著变化。该文从TET家族蛋白展开,介绍TET蛋白的结构、功能及作用机制以及多种人类肿瘤中丁E丁家族基因与5-hmC水平的相关性及其对肿瘤发生发展、诊断预后等临床意义的研究进展。     

Tetrandrine cytotoxicity and its dual effect on oxidative stress-induced apoptosis through modulating cellular redox states in Neuro 2a mouse neuroblastoma cells

Tetrandrine (TET), a plant alkaloid, is known primarily as a non-selective Ca(2+) channel blocker. On the contrary to the cytoprotective effect on ischemia/reperfusion injury, TET has also been reported to cause cytotoxicity. In this study, we wished to understand the apparently disparate effects of this potential drug and thus investigated molecular mechanisms on proliferation and apoptosis and its effect on oxidative stress-induced apoptosis in Neuro 2a mouse neuroblastoma cells. We showed that TET, at high concentrations, induced cell cycle arrest and apoptosis through oxidative stress with following observations. Firstly, 10 microM TET elevated the reactive oxygen species (ROS) level and accordingly depleted glutathione (GSH) content. Secondly, pretreatment with antioxidants (NAC or GSH) protected cells from TET-induced apoptosis. We also demonstrated that treatment with 10 microM TET caused not only induction of p53, p21(waf1), and Bax, but also nuclear translocation of p53 and hypo-phosphorylation of pRb concurrently. Our important finding is that the concentration-dependent dual effect of TET, either inhibiting or promoting cell death induced by H(2)O(2) was observed, probably through regulating redox balance, which was well reflected on the GSH content in each condition. Besides, inhibition of Ca(2+) influx protected cells from H(2)O(2)-induced apoptosis even in the presence of 10 microM TET. Taken together, our data suggest that TET regulation of cellular redox states may play a major role in its dual action of cytotoxicity and cytoprotection.     

β2SP/TET2 complex regulates gene 5hmC modification after cerebral ischemia

βII spectrin (β2SP) is encoded by Sptbn1 and is involved in the regulation of various cell functions. β2SP contributes to the formation of the myelin sheath, which may be related to the mechanism of neuropathy caused by demyelination. As one of the main features of cerebral ischemia, demyelination plays a key role in the mechanism of cerebral ischemia injury. Here, we showed that β2SP levels were increased, and this molecule interacted with TET2 after ischemic injury. Furthermore, we found that the level of TET2 was decreased in the nucleus when β2SP was knocked out after oxygen and glucose deprivation (OGD), and the level of 5hmC was reduced in the OGD+β2SP KO group. In contrast, the expression of β2SP did not change in TET2 KO mice. In addition, the 5hmC sequencing results revealed that β2SP can affect the level of 5hmC, the differentially hydroxymethylated region (DhMR) mainly related with the Calcium signalling pathway, cGMP-PKG signalling pathway, Wnt signalling pathway and Hippo signalling pathway. In summary, our results suggest that β2SP could regulate the gene 5hmC by interacted with TET2 and will become a potential therapeutic target for ischemic stroke.     

TET2在帕金森病细胞模型中的表达及机制研究

目的:迄今为止,帕金森病(PD)发生的分子机制尚未完全阐明,本研究旨在体外细胞模型中寻找PD新型表观遗传标志物,探索其发病机制。方法:本次研究使用的细胞为神经母细胞瘤细胞系SH-SY5Y。首先,我们用CCK-8检测细胞活力,选取合适浓度的MPP+构建PD细胞损伤模型。再用PBS和MPP+分别处理SH-SY5Y细胞,用RT-qPCR检测了几个甲基化酶与去甲基化酶DNMT1,DNMT3A,DNMT3B及TET1, TET2, TET3的mRNA的表达水平,并用蛋白印迹检测TET2蛋白水平,免疫荧光检测了TET2蛋白定位。进一步用慢病毒转染SH-SY5Y细胞敲低TET2后,检测细胞增殖。结果:本研究发现,MPP+对SH-SY5Y细胞增殖的抑制具有时间与浓度依赖性,我们最终选择2.5 mM MPP+作为后续的细胞处理浓度。与对照组相比,MPP+处理细胞TET2的mRNA及蛋白水平表达均增加,且蛋白进入细胞核增加;同时发现,敲低TET2表达可以延缓MPP+对SH-SY5Y细胞增殖的抑制作用。结论:在当前的研究中,我们报道了TET2蛋白可能是PD新型的表观遗传学标志物,提示我们将来也许可以使用TET2抑制剂来治疗PD,因此本研究有可能为PD提供新的治疗方向和靶点。     

哺乳动物DNA甲基化修饰的动态调控机制

DNA甲基化是最主要的表观遗传修饰之一,主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶。哺乳动物DNA甲基化由从头DNA甲基转移酶DNMT3A/3B在胚胎发育早期建立。细胞分裂过程中甲基化模式的维持由DNA甲基转移酶DNMT1实现。TET家族蛋白氧化5-甲基胞嘧啶成为5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,从而起始DNA的去甲基化过程。这些DNA甲基化修饰酶精确调节DNA甲基化的动态过程,在整个生命发育过程中发挥重要作用,其失调也与多种疾病发生密切相关。本文对近年来DNA甲基化修饰酶的结构与功能研究进行讨论。