5-hydroxymethylcytosine in DNA repair: A new player or a red herring?

Active DNA demethylation performed by ten-eleven translocation (TET) enzymes produces 5-hydroxymethylcytosines, 5-formylcytosines, and 5-carboxylcytosines. Recent observations suggest that 5-hydroxymethylcytosine is a stable epigenetic mark rather than merely an intermediate of DNA demethylation. However, the clear functional role of this new epigenetic player is elusive. The contribution of 5-hydroxymethylation to DNA repair is being discussed currently. Recently, Jiang and colleagues have demonstrated that DNA damage response-activated ATR kinase phosphorylates TET3 in mammalian cells and promotes DNA demethylation and 5-hydroxymethylcytosine accumulation. Moreover, TET3 catalytic activity is important for proper DNA repair and cell survival. Here, we discuss recent studies on the potential role of 5-hydroxymethylation in DNA repair and genome integrity maintenance.     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

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.     

Application of induced pluripotency in cancer studies

As soon as induced pluripotent stem cells (iPSCs) reprogramming of somatic cells were developed, the discovery attracted the attention of scientists, offering new perspectives for personalized medicine and providing a powerful platform for drug testing. The technology was almost immediately applied to cancer studies. As presented in this review, direct reprogramming of cancer cells with enforced expression of pluripotency factors have several basic purposes, all of which aim to explain the complex nature of cancer development and progression, therapy-resistance and relapse, and ultimately lead to the development of novel anti-cancer therapies. Here, we briefly present recent advances in reprogramming methodologies as well as commonalities between cell reprogramming and carcinogenesis and discuss recent outcomes from the implementation of induced pluripotency into cancer research.     

外周血细胞重编程为诱导性多潜能干细胞的研究进展

诱导性多潜能干细胞(iPSCs)是指分化细胞中导入特定转录因子后逆转恢复到类似胚胎干细胞的具有自我更新、多向分化等潜能的一类细胞。诱导疾病特异性iPSCs是疾病机理、再生医学等领域的研究热点。目前,人iPSCs供体细胞主要来源于皮肤成纤维细胞,需要组织活检、体外增殖等繁琐过程。利用外周血细胞(peripheral blood cells)成功诱导iPSCs,具有取材方便、诱导快速等优点,将极大地促进iPSCs研究。该文在介绍iPSCs诱导方法的基础上,重点阐述了从小鼠B细胞、T细胞,人脐带血细胞,到人外周血细胞重编程为iPSCs的研究进展,分析了该技术的特点和可能存在的问题,并进行了前景展望。     

Reprogramming cancer cells to pluripotency: An experimental tool for exploring cancer epigenetics

The epigenetic marks displayed by a cancer cell originate from two separate processes: The most prominent epigenetic signatures are associated with the cell of origin, i.e., the lineage and cell type identity imposed during development. The second set comprises those aberrant cancer-specific epigenetic marks that appear during tumor initiation or subsequent malignant progression. These are generally thought to associate with tumor-promoting pathways. As biochemical pathways regulating epigenetic mechanisms are potentially “druggable” and reversible, there is considerable interest in defining their roles in tumor genesis and growth, as they may represent therapeutic targets for treatment of human neoplasias.¹ However, despite the potential importance of epigenetic modifications in human cancer, it has been difficult to determine when, where and how epigenetic disruptions occur, and if they have important functional roles in sustaining the malignant state.     

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

Metabotropic glutamate receptor 5 responses dictate differentiation of neural progenitors to NMDA‐responsive cells in fragile X syndrome

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)−3,5‐dihydroxyphenylglycine (DHPG) are augmented, and calcium‐dependent mGluR5‐mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl)‐pyridine (MPEP) prevents an abnormal clustering of DHPG‐responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron‐like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG‐responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS‐specific increase in the differentiation of cells responsive only to N ‐methyl‐d ‐aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type‐dependent and species‐specific differences of basal progenitors may modify effects of MPEP on the cortical development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Specificity of MLL1 and TET3 CXXC domains towards naturally occurring cytosine modifications

CXXC domains have traditionally been considered as CpG specific DNA binding domains that are repelled by cytosine modifications. This view has recently been challenged by the demonstration that CXXC domain of TET3 has relaxed sequence specificity and binds with the highest affinity to symmetric DNA duplex containing 5caCpG. Here, we present a comparative analysis of the MLL1-CXXC and TET3-CXXC sequence specificity and tolerance to cytosine modifications (5-methyl, 5-hydroxymethyl, 5-formyl, 5-carboxyl) in CpG and non-CpG context. For the first time, we take into consideration possible interference from cytosine bases elsewhere in the sequence. We show that despite similar overall structure, MLL1-CXXC has greater sequence and modification specificity than TET3-CXXC. MLL1-CXXC is specific only for CpG and does not tolerate any cytosine modifications. In contrast, TET3-CXXC does not require the CpG context of cytosine bases. Methyl-, formyl- and carboxyl-modifications are tolerated by TET3-CXXC, but only preceding G. Based on our and other data we propose a parsimonious model of MLL1-CXXC and TET3-CXXC DNA binding. This model explains why the binding of modified DNA duplexes by TET3-CXXC requires in some cases a register shift and is therefore context-dependent.     

Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Pluripotent Stem Cells for Uncovering the Role of Mitochondria in Human Brain Function and Dysfunction

Mitochondrial dysfunctions are a known pathogenetic mechanism of a number of neurological and psychiatric disorders. At the same time, mutations in genes encoding for components of the mitochondrial respiratory chain cause mitochondrial diseases, which commonly exhibit neurological symptoms. Mitochondria are therefore critical for the functionality of the human nervous system. The importance of mitochondria stems from their key roles in cellular metabolism, calcium handling, redox and protein homeostasis, and overall cellular homeostasis through their dynamic network. Here, we describe how the use of pluripotent stem cells (PSCs) may help in addressing the physiological and pathological relevance of mitochondria for the human nervous system. PSCs allow the generation of patient-derived neurons and glia and the identification of gene-specific and mutation-specific cellular phenotypes via genome engineering approaches. We discuss the recent advances in PSC-based modeling of brain diseases and the current challenges of the field. We anticipate that the careful use of PSCs will improve our understanding of the impact of mitochondria in neurological and psychiatric disorders and the search for effective therapeutic avenues.     

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.