Loss of 5-Hydroxymethylcytosine Is an Independent Unfavorable Prognostic Factor for Esophageal Squamous Cell Carcinoma
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Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxylcytosine, which result in genomic DNA demethylation. It was reported that 5-hmC levels were decreased in a variety of cancers and could be regarded as an epigenetic hallmark of cancer. In the present study, 5-hmC levels were detected by immunohistochemistry (IHC) in 173 esophageal squamous cell carcinoma (ESCC) tissues and 91 corresponding adjacent non-tumor tissues; DNA dot blot assays were used to detect the 5-hmC level in another 50 pairs of ESCC tissues and adjacent non-tumor tissues. In addition, the mRNA level of TET1, TET2 and TET3 in these 50 pairs of ESCC tissues was detected by real-time PCR. The IHC and DNA dot blot results showed that 5-hmC levels were significantly lower in ESCC tissues compared with corresponding adjacent non-tumor tissues (P = 0.029). TET2 and TET3 expression was also significantly decreased in tumor tissues compared with paired non-tumor tissues (TET2, P < 0.0001; TET3, P = 0.009), and the decrease in 5-hmC was significantly associated with the downregulation of TET2 expression (r = 0.405, P = 0.004). Moreover, the loss of 5-hmC in ESCC tissues was significantly associated with poor overall survival among patients with ESCC (P = 0.043); multivariate Cox regression analysis showed that the loss of 5-hmC in ESCC tissues was an independent unfavorable prognostic indicator for patients with ESCC (HR = 1.569, P = 0.029). In conclusion, 5-hmC levels were decreased in ESCC tissues, and the loss of 5-hmC in tumor tissues was an independent unfavorable prognostic factor for patients with ESCC.Keywords:
5-Hydroxymethylcytosine
DNA demethylation
Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in mammalian neurons, our knowledge of its roles in terminal differentiation or as an intermediate in active DNA demethylation is incomplete. We report high-resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and histone marks in developing postmitotic Purkinje cells (PCs) in Mus musculus. Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of genes that lose both 5-methylcytosine (5mC) and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic PCs prevents loss of 5mC and 5hmC in regulatory domains and gene bodies, and hinders transcriptional and epigenetic developmental transitions. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular properties of adult PCs require continued oxidation of 5mC to 5hmC during the final phases of differentiation.At birth, the mammalian brain contains tens of billions of neurons. Although the number does not increase much as the animal grows, there are many dramatic changes to their size and structure. These changes allow the neurons to communicate with one another, develop into networks, and learn the tasks of the adult brain. One way that these changes occur is by the accumulation of chemical marks on each neuron’s DNA that help dictate which genes switch on, and which turn off. One of the most common ways that DNA can be marked is through the addition of a chemical group called a methyl group to one of the four DNA bases, cytosine. This process is called methylation. When methylation occurs, cytosine becomes 5-methylcytosine, or 5mC for short. In 2009, researchers found another modification present in the DNA in the brain: 5-hydroxymethylcytosine, or 5hmC. This modification appears when a group of proteins called the Tet hydroxylases turn 5mC into 5hmC. Converting 5mC to 5hmC normally helps cells remove marks on their DNA before they divide and expand. This is important because the newly generated cells need to be able to accumulate their own methylation marks to perform their roles properly. However, neurons in the brain accumulate 5hmC after birth, when the cells are no longer dividing, indicating that 5hmC may be required for the neurons to mature. Stoyanova et al. set out to determine whether mouse neurons need 5hmC to get their adult characteristics by tracking the chemical changes that occur in DNA from birth to adulthood. Some of the mice they tested produced 5hmC normally, while others lacked the genes necessary to make the Tet proteins in a specific class of neurons, preventing them from converting 5mC to 5hmC as they differentiate. The results reveal that neurons do not mature properly if 5hmC is not produced continuously following the first week of life. This is because neurons need to have the right genes switched on and off to differentiate correctly, and this only happens when 5hmC accumulates in some genes, while 5hmC and 5mC are removed from others. The data highlight the role of the Tet proteins, which convert 5mC into 5hmC, in preparing the marks for removal and demonstrate that active removal of these marks is essential for neuronal differentiation. Given the role of 5hmC in the development of neurons, it is possible that problems in this system could contribute to brain disorders. Further studies aimed at understanding how cells control 5hmC levels could lead to new ways to improve brain health. Research has also shown that if dividing cells lose the ability to make 5hmC, they can become cancerous. Future work could explain more about how and why this happens.
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Abstract Dynamic DNA methylation is a prerequisite for many developmental processes and maintenance of cellular integrity. In mammals however, mechanisms of active DNA demethylation have for long been elusive. The discovery of the ten-eleven translocation (Tet) family of enzymes that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) provided new means by which DNA methylation could actively be reversed. This review focuses on the possible mechanisms of DNA demethylation via Tet proteins and their metabolites 5hmC, 5fC and 5caC. Additionally, it discusses the roles of the three Tet protein family members Tet1, Tet2 and Tet3 as developmental regulators, probably in part independent of their enzymatic activity. By contrast, recent evidence suggests a function of 5hmC as an epigenetic mark on its own, going beyond the expectation of only acting as an intermediate in an active DNA demethylation pathway.
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DNA demethylation
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The recently re-discovered 5-hydroxymethylcytosine (5hmC) is established as the sixth base and found in various mammalian tissues. Although the content of 5hmC is much lower than that of the best known epigenetic mark 5-methylcytosine (5mC), 5hmC plays key roles in nuclear reprogramming, regulates the gene activity, and initiates the DNA demethylation in mammals. The formation of 5hmC results from the oxidation of 5mC in genomic DNA, which is mediated by Tet (ten eleven translocation) family dioxygenases. The Tet-mediated 5mC oxidation has just been identified. The 5hmC formation can cause DNA replication-dependent and passive DNA demethylation, meanwhile, it can also induce active DNA demethylation by further oxidation forming 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be removed by base excision repair. Essentially, the discovery of 5fC and 5caC indicates the occurrence of active DNA demethylation mechanisms. The 5fC and 5caC have been detected in genomic DNA of mouse embryonic stem cells, but 5hmC has been found in various tissues. The 5hmC abundance in genomic DNA of certain tumors is found to be associated with the tumor process. However, the genome-wide distribution and sequence selectivity of 5hmC are not known yet. 5hmC cannot be dis- criminated from 5mC by the well-known bisulfite sequencing. Therefore, it is essential to develop new methods and tech- nologies for detecting and sequencing 5hmC. To this purpose, the two or more of the technologies for modification of 5hmC by glucosylation or chemical labeling, restriction enzymatic digestion, affinity capture, liquid chromatography and mass spectrometry, and bisulfite sequencing are combined. The further combination with next-generation high-throughput DNA sequencing technologies may provide genome-wide information. The single molecule real time DNA sequencing is also of choice to detect 5hmC at the gene level or the genome level. Recently, oxidative bisulfite sequencing was for the first time developed for quantitative mapping of 5hmC in genomic DNA at single base resolution. The genome-wide 5hmC sequencing at single base resolution is also reported. Here we briefly review and discuss the detection and sequencing technologies for 5hmC analysis. Keywords 5-hydroxymethylcytosine; 5-methylcytosine; DNA sequencing
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Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining. We were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes. In conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development.
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DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation. Here, we used whole-genome bisulfite and transcriptome sequencing to characterize the underlying mechanisms. Our results uncover the hypermethylation of DNA methylation canyons as the genomic key feature of Tet1/Tet2 double-knockout mouse embryonic fibroblasts. Canyon hypermethylation coincided with disturbed regulation of associated genes, suggesting a mechanistic explanation for the observed Tet-dependent differentiation defects. Based on these results, we propose an important regulatory role of Tet-dependent DNA demethylation for the maintenance of DNA methylation canyons, which prevents invasive DNA methylation and allows functional regulation of canyon-associated genes.
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Epigenetic processes, such as DNA methylation and other chromatin modifications, are believed to be largely responsible for establishing a reduced capacity for growth in the mature nervous system. Ten-eleven translocation methylcytosine dioxygenase 3 (Tet3)-, a member of the Tet gene family, plays a crucial role in promoting injury-induced DNA demethylation and expression of regeneration-associated genes in the peripheral nervous system. Here, we encapsulate Tet3 protein within a clinically tolerated poly(lactide-co-glycolide) microsphere system. Next, we show that Tet3-loaded microspheres are internalized into mHippoE-18 embryonic hippocampal cells. We compare the outgrowth potential of Tet3 microspheres with that of commonly used nerve growth factor (NGF)-loaded microspheres in an in vitro injury model. Tet3-containing microspheres increased levels of nuclear 5-hydroxymethylcytosine indicating active demethylation and outperformed NGF-containing microspheres in measures of neurite outgrowth. Our results suggest that encapsulated demethylases may represent a novel avenue to treat nerve injuries.
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The pursuit of DNA demethylation has a colorful history, but it was not until 2009 that the stars of this story, the Ten-eleven-translocation (Tet) family of proteins, were really identified. Tet proteins convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine and 5-cyboxycytosine by Tet proteins to achieve DNA demethylation. Recent studies have revealed that 5hmC-mediated DNA demethylation can play essential roles in diverse biological processes, including development and diseases. Here, we review recent discoveries in 5hmC-mediated DNA demethylation in the context of stem cells and development.
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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.
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