Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.     

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.     

Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis

DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but not in parthenogenetically activated embryos. Expression of Ten-Eleven Translocation 1 (TET1) correlates with this conversion. Expression of 5mC gradually decreases until the morula stage; it is only expressed in the inner cell mass, but not trophectoderm regions of IVV and IVF blastocysts. Expression of 5mC in SCNT embryos is ectopically distinct from that observed in IVV and IVF embryos. In addition, 5hmC expression was similar to that of 5mC in IVV cleavage-stage embryos. Expression of 5hmC remained constant in IVF and SCNT embryos, and was evenly distributed among the inner cell mass and trophectoderm regions derived from IVV, IVF, and SCNT blastocysts. Ten-Eleven Translocation 3 was highly expressed in two-cell embryos, whereas TET1 and TET2 were highly expressed in blastocysts. These data suggest that TET1-catalyzed 5hmC may be involved in active DNA demethylation in porcine early embryos. In addition, 5mC, but not 5hmC, participates in the initial cell lineage specification in porcine IVV and IVF blastocysts. Last, SCNT embryos show aberrant 5mC and 5hmC expression during early porcine embryonic development.     

A Zebrafish Model of Myelodysplastic Syndrome Produced through tet2 Genomic Editing

The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2^m/m (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2^m/m mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2^m/m zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells.     

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.     

Mouse olfactory bulb methylome and hydroxymethylome maps reveal noncanonical active turnover of DNA methylation

Hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes presents a particular regulatory mechanism in the mammalian brain. However, although methylation and hydroxymethylation of cytosines in non-CpG contexts have been reported, these mechanisms remain poorly understood. Here, we applied TAB-seq and oxBS-seq selectively to detect 5hmC and 5mC at base resolution in olfactory bulb derived from female mice. We found that active turnover of 5mC to 5hmC occurred in both CpG and non-CpG contexts. Strikingly, we identified a different sequence preference for 5mC and 5hmC in a CH context, in which H = A, C, or T, TNCA/TC for 5mC and NNCA/T/CN for 5hmC. More importantly, we found that genes showing 5mC to 5hmC turnover showed only limited overlap in CpG and CH contexts, and that olfactory receptor genes were marked with higher turnover of 5mC to 5hmC in non-CpG context. Collectively, we identified an unexpected sequence preference for non-CpG hydroxymethylation and distinct target genes regulated by the turnover of 5mC to 5hmC in CpG and CH contexts.     

Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase

In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7–10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.     

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.     

The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.     

5-羟甲基胞嘧啶及其TET氧合酶在神经系统发育和相关疾病中的研究进展

神经系统的正常发育是多种因素相互协调作用的结果,一旦特定因素失衡将引起相关疾病的发生。近年来不断有研究发现,DNA去甲基化过程的一类中间产物5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)作为一种新的表观遗传标记,在神经系统中高水平分布,并参与认知、记忆等重要的神经功能。5hmC的形成由氧合酶家族分子(ten-eleven translocation protein, TET)催化,在多种神经系统相关疾病中,5hmC水平和TETs分子的表达都发生改变,提示TET-5hmC表观遗传机制在复杂的神经系统发生发展过程中发挥了重要的调控作用。此外,作为基因表达调控的DNA标记物,5hmC的基因定位与基因表达水平的关系也是重要的研究方向。本文就近年来5hmC和TET家族蛋白分子在神经系统发育和相关疾病方面的重要研究发现进行了综述总结,希望为相关领域研究人员深入开展研究提供重要的思路,并为相关疾病设计治疗策略提供理论支持。     

TET蛋白：一个新的DNA修饰酶家族

DNA的胞嘧啶（C）5-甲基化是一种重要的表观修饰,它参与基因调节、基因组印记、X-染色体失活、重复序列抑制和癌症发生等过程. 5-甲基胞嘧啶（5mC）可被TET (ten-eleven translocation)蛋白家族进一步转化为5-羟甲基胞嘧啶（5hmC）,该过程是DNA去甲基化的1个必要阶段. 5hmC可在活性转录基因起始位点和Polycomb抑制基因启动子延伸区域富集.TET蛋白包括3个成员TET1、TET2和TET3,均属于α-酮戊二酸和Fe²⁺依赖的双加氧酶,其催化涉及氧化过程.小鼠Tet1在胚胎干细胞发育中拥有双重作用,即促进全能因子的转录,又参与发育调节因子的抑制.人TET蛋白的破坏与造血系统肿瘤相关,如在骨髓增生性疾病/肿瘤存在频繁的TET2基因突变.TET蛋白和5hmC的研究为DNA甲基化/去甲基化及其生物学功能提供了新的视点.