Role Of DNA Demethylation Research Articles

AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation.

Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The TET2 gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism. To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting. The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium. Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.

Association of tet methylcytosine dioxygenase 2 and 5-hydroxymethylcytosine in endometrioid adenocarcinoma and its clinical significance

BackgroundAberrant DNA methylation is a vital molecular alteration commonly detected in type I endometrial cancers (EC), and tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine (5hmC) play significant roles in DNA demethylation. However, little is known about the function and correlation of TET2 and 5hmC co-expressed in EC. This study intended to investigate the clinical significance of TET2 and 5hmC in EC.MethodsThe levels of TET2 and 5hmC were detected in 326 endometrial tissues by immumohistochemistry, and the correlation of their level was detected by Pearson analysis. The association between the levels of TET2 and 5hmC and clinicopathologic characteristics was analyzed. Prognostic value of TET2 and 5hmC was explored by Kaplan–Meier analysis. The Cox proportional hazard regression model was used for univariate and multivariate analyses.ResultsBased on the analysis results, TET2 protein level was positively correlated with 5hmC level in EC tissues (r = 0.801, P < 0.001). TET2+5hmC+ (high TET2 and high 5hmC) association was significantly associated with well differentiation, myometrial invasion, negative lymph node metastasis, and tumor stage in EC. Association of TET2 and 5hmC was confirmed as a prognostic factor (HR = 2.843, 95%CI = 1.226–3.605, P = 0.007) for EC patients, and EC patients with TET2−5hmC− level had poor overall survival.ConclusionsIn summary, the association of TET2 and 5hmC was downregulated in EC tissues, and may be a potential poor prognostic indicator for EC patients. Combined detection of TET2 and 5hmC may be valuable for the diagnosis and prognosis of EC.

The role of DNA demethylation in liver to pancreas transdifferentiation

BackgroundInsulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation.ResultsThe transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process.ConclusionsTransdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation.

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.

DNA demethylation affects imprinted gene expression in maize endosperm

BackgroundDNA demethylation occurs in many species and is involved in diverse biological processes. However, the occurrence and role of DNA demethylation in maize remain unknown.ResultsWe analyze loss-of-function mutants of two major genes encoding DNA demethylases. No significant change in DNA methylation has been detected in these mutants. However, we detect increased DNA methylation levels in the mutants around genes and some transposons. The increase in DNA methylation is accompanied by alteration in gene expression, with a tendency to show downregulation, especially for the genes that are preferentially expressed in endosperm. Imprinted expression of both maternally and paternally expressed genes changes in F1 hybrid with the mutant as female and the wild-type as male parental line, but not in the reciprocal hybrid. This alteration in gene expression is accompanied by allele-specific DNA methylation differences, suggesting that removal of DNA methylation of the maternal allele is required for the proper expression of these imprinted genes. Finally, we demonstrate that hypermethylation in the double mutant is associated with reduced binding of transcription factor to its target, and altered gene expression.ConclusionsOur results suggest that active removal of DNA methylation is important for transcription factor binding and proper gene expression in maize endosperm.

Prognostic Utility of CpG Island Hypermethylated Phenotype in Early-Stage Invasive Primary Melanomas

In their article in the Journal of Investigative Dermatology, Conway etal. (2021) show the significance of the CpG island methylator phenotype (CIMP) subclass in early-stage invasive primary melanomas, particularly in the lentigo maligna melanoma histological subtype. CIMP melanomas had reduced melanoma-specific survival compared with intermediate and low methylation subtypes. This study emphasizes the importance of epigenomic changes in early-stage primary tumors that are associated with poor prognosis.

Deoxyribonucleic Acid 5-Hydroxymethylation in Cell-Free Deoxyribonucleic Acid, a Novel Cancer Biomarker in the Era of Precision Medicine.

Aberrant methylation has been regarded as a hallmark of cancer. 5-hydroxymethylcytosine (5hmC) is recently identified as the ten-eleven translocase (ten-eleven translocase)-mediated oxidized form of 5-methylcytosine, which plays a substantial role in DNA demethylation. Cell-free DNA has been introduced as a promising tool in the liquid biopsy of cancer. There are increasing evidence indicating that 5hmC in cell-free DNA play an active role during carcinogenesis. However, it remains unclear whether 5hmC could surpass classical markers in cancer detection, treatment, and prognosis. Here, we systematically reviewed the recent advances in the clinic and basic research of DNA 5-hydroxymethylation in cancer, especially in cell-free DNA. We further discuss the mechanisms underlying aberrant 5hmC patterns and carcinogenesis. Synergistically, 5-hydroxymethylation may act as a promising biomarker, unleashing great potential in early cancer detection, prognosis, and therapeutic strategies in precision oncology.

Loss of ten-eleven translocation 1 (TET1) expression as a diagnostic and prognostic biomarker of endometrial carcinoma.

Endometrial carcinoma (EC) is the most common gynecological cancer. However, there is currently no routinely used biomarker for differential diagnosis of malignant and premalignant endometrial lesions. Ten-eleven translocation (TET) proteins, especially TET1, were found to play a significant role in DNA demethylation, via conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1, 5-mC, and 5-hmC expression profiles in endometrial carcinogenesis are currently unclear. We conducted a hospital-based retrospective review of the immunohistochemical expression of TET1, 5-mC, and 5-hmC in 181 endometrial samples. A “high” TET1 and 5-hmC expression score was observed in all cases of normal endometrium (100.0% and 100.0%, respectively) and in most samples of endometrial hyperplasia without atypia (90.9% and 78.8%, respectively) and atypical hyperplasia (90.6% and 93.8%, respectively), but a “high” score was found in only less than half of the EC samples (48.8% and 46.5%, respectively). The TET1 and 5-hmC expression scores were significantly higher in normal endometrium and premalignant endometrial lesions than in ECs (p < 0.001). A “high” 5-mC expression score was observed more frequently for ECs (81.4%) than for normal endometrium (40.0%), endometrial hyperplasia without atypia (51.5%), and atypical hyperplasia (53.1%) (p < 0.001). We also found that TET1 mRNA expression was lower in ECs compared to normal tissues (p = 0.0037). TET1 immunohistochemistry (IHC) scores were highly proportional to the TET1 mRNA levels and we summarize that the TET1 IHC scoring can be used for biomarker determinations. Most importantly, a higher TET1 score in EC cases was associated with a good overall survival (OS) rate, with a hazard ratio (HR) of 0.31 for death (95% confidence interval: 0.11–0.84). Our findings suggest that TET1, 5-mC, and 5-hmC expression is a potential histopathology biomarker for the differential diagnosis of malignant and premalignant endometrial lesions. TET1 is also a potential prognostic marker for EC.

SAR insights into TET2 catalytic domain inhibition: Synthesis of 2-Hydroxy-4-Methylene-pentanedicarboxylates

The TET (Ten-Eleven Translocation) dioxygenase enzyme family comprising 3 members, TET1-3, play key roles in DNA demethylation. These processes regulate transcription programs that determine cell lineage, survival, proliferation, and differentiation. The impetus for our investigations described here is derived from the need to develop illuminating small molecule probes for TET enzymes with cellular activity and specificity. The studies were done so in the context of the importance of TET2 in the hematopoietic system and the preponderance of loss of function somatic TET2 mutations in myeloid diseases. We have identified that 2-hydroxy-4-methylene-pentanedicarboxylic acid 2a reversibly competes with the co-substrate α-KG in the TET2 catalytic domain and inhibits the dioxygenase activity with an IC50 = 11.0 ± 0.9 μM at 10 μM α-KG in a cell free system and binds in the TET2 catalytic domain with Kd = 0.3 ± 0.12 μM.

Insights into the Role of Transcriptional Gene Silencing in Response to Herbicide-Treatments in Arabidopsis thaliana.

Herbicide resistance is broadly recognized as the adaptive evolution of weed populations to the intense selection pressure imposed by the herbicide applications. Here, we tested whether transcriptional gene silencing (TGS) and RNA-directed DNA Methylation (RdDM) pathways modulate resistance to commonly applied herbicides. Using Arabidopsis thaliana wild-type plants exposed to sublethal doses of glyphosate, imazethapyr, and 2,4-D, we found a partial loss of TGS and increased susceptibility to herbicides in six out of 11 tested TGS/RdDM mutants. Mutation in REPRESSOR OF SILENCING 1 (ROS1), that plays an important role in DNA demethylation, leading to strongly increased susceptibility to all applied herbicides, and imazethapyr in particular. Transcriptomic analysis of the imazethapyr-treated wild type and ros1 plants revealed a relation of the herbicide upregulated genes to chemical stimulus, secondary metabolism, stress condition, flavonoid biosynthesis, and epigenetic processes. Hypersensitivity to imazethapyr of the flavonoid biosynthesis component TRANSPARENT TESTA 4 (TT4) mutant plants strongly suggests that ROS1-dependent accumulation of flavonoids is an important mechanism for herbicide stress response in A. thaliana. In summary, our study shows that herbicide treatment affects transcriptional gene silencing pathways and that misregulation of these pathways makes Arabidopsis plants more sensitive to herbicide treatment.

Ascorbic acid during the suckling period is required for proper DNA demethylation in the liver

Ascorbic acid (AA, vitamin C) serves as a cofactor for ten-eleven translocation (TET) enzymes and induces DNA demethylation in vitro. However, its role in DNA demethylation in vivo remains unclear. We previously reported that DNA demethylation in the mouse liver was enhanced during the suckling period. Therefore, we hypothesized that DNA demethylation is enhanced in an AA-dependent manner during the suckling period. To examine our hypothesis, we employed wild-type (WT) mice, which synthesize AA, and senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA, and analyzed the DNA methylation status in the livers of offspring in both the suckling period and adulthood. SMP30/GNL KO offspring showed DNA hypermethylation in the liver possibly due to low plasma and hepatic AA levels during the suckling period despite the administration of rescue-dose AA to dams. Furthermore, DNA hypermethylation of the fibroblast growth factor 21 gene (Fgf21), a PPARα target gene, persisted into adulthood. In contrast, a high-dose AA administration to SMP30/GNL KO dams during the lactation period restored DNA demethylation in the livers of offspring. Even though a slight increase was observed in plasma AA levels with the administration of rescue-dose AA to WT dams during the gestation and lactation periods, DNA demethylation in the livers of offspring was minimally enhanced. The present results demonstrate that AA intake during the suckling period is required for proper DNA demethylation in the liver.

Tet2 regulates Barx2 expression in undifferentiated and early differentiated mouse embryonic stem cells

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. In developing embryos, TET2 are upregulated during pre-implantation development, and significantly expressed in the trophectoderm and inner cell mass. In this study, we identified Barx2 as a new target of Tet2. Tet2 bound and demethylated the promoter of Barx2 in mouse embryonic stem cells (mESCs) to maintain the expression of Barx2. During mESC differentiation, Tet2 bound the promoter of Barx2 in day 4 embryonic bodies but not in day 8 EBs. However, Barx2 expression remained unchanged. Thus, Tet2 functioned as a demethylase and maintained the expression of Barx2 in undifferentiated and early differentiated mESCs.

46 Presence of porcine TET3L isoform in oocytes: Potential involvement in the DNA demethylation process

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.

DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis

ABSTRACTDNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:β-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable ‘quadruple’ demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.

SUN-043 Epigenetic Regulation of Aldosterone Synthase Gene by Potassium

Purpose: DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We reported that angiotensin II could change the DNA methylation status around transcription factor binding sites and a transcription start site (TSS) and activate expression of the aldosterone synthase gene (CYP11B2). In order to clarify the effect of potassium on the methylation status of CYP11B2 H295R cells were treated with high concentration of potassium and the methylation status of CYP11B2 was examined. Methods: H295R cells were treated with 16mM potassium for 7 days and observed the dynamic changes in DNA methylation patterns of the CYP11B2 promoter for 28 days. The gene expression of CYP11B2 was measured by real time quantitative PCR. Isolated DNAs from H295R cells were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions. Non-treated cells served as a control. Human CYP11B2 ELISA, chromatin accessibility by real-time PCR (CHART-PCR), DNA methylation and demethylation activity assay were done as previously reported. Results: DNA demethylation at CpG1 (Ad1) was detected within 2 days after stimulation. DNA at CpG2 (Ad5) and CpG3 and DNA around a transcription start site (CpG-1/-2) were demethylated at day 4 in cells treated with potassium. CYP11B2 mRNA levels in cells treated with potassium were significantly higher than those in non-treated cells at days 7 and 9. Similar changes were observed in the levels of CYP11B2 protein. CHART-PCR revealed that potassium treatment increased chromatin accessibility around Ad1 at days 2, 4, 7, 9 and 11, with accessibility increasing from 67% to 70%. Potassium treatment significantly reduced DNA methylation activity at days 7 and 9. Demethylation activity was not affected by potassium. Conclusion: Our data indicated that binding of transcription factors initiates chromatin relaxation and transcription, which are followed by DNA demethylation around transcription factor binding sites and a TSS in the CYP11B2 promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of CYP11B2 expression from an inactive to an active state.

DNA Hydroxymethylation Regulates Beta-Cell Maturation and Expansion

Epigenetic mechanisms play a central role in governing functional beta-cell mass. In particular, the methylation of DNA to generate 5-methylcytosine (5mC), directed by DNA methyltransferases, has been shown to regulate the establishment and maintenance of beta-cell identity and function. While the contribution of DNA methylation to beta-cell homeostasis and diabetes pathogenesis has been studied in detail, not much is known about the role of DNA demethylation in beta-cells. Oxidative conversion of 5mC to 5-hydroxymethylcytosine (5hmC) by Ten Eleven Translocation (Tet) enzymes constitutes a key step towards active DNA demethylation. In the present study, we provide evidence that the 5hmC landscape is dynamic, and undergoes specific changes during embryonic and postnatal islet development, islet adaptation, and in diabetes. We report that the Pdx1-positive pancreatic progenitors are marked by very low 5hmC, and beta-cell differentiation and functional maturation is accompanied by a progressive increase in 5hmC. However, under conditions during adaptive beta-cell expansion as well as in diabetes 5hmC levels decline in part due to the oxidative stress. These changes in 5hmC pattern are conserved in mouse and human pancreas, and are accompanied by the changes in Tet2 levels. We show that deletion of Tet2 in the pancreatic lineage results in improved glucose tolerance and beta-cell function. Together, our data show the importance of Tet2-dependent 5hmC patterning in maintaining a differentiated beta-cell phenotype, and highlight the remodeling of beta-cell epigenetic landscape as a key component of diabetes pathogenesis. Disclosure T. Gurlo: None. S. Georgia: None. S. Dhawan: None.

Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization

BackgroundThe estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specific enhancer regions in response to β-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood.ResultsUsing ChIP-Seq we generate a global profile of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifications indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we find that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells.ConclusionsTaken together we find that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an effective therapeutic target.

Effect of TET inhibitor on bovine parthenogenetic embryo development.

DNA demethylation catalysed by the ten-eleven translocation (TET) protein is an important step during extensive global epigenetic reprogramming in mammals. However, whether TET proteins play a key role in DNA demethylation during the development of bovine pre-implanted embryos is still unclear. In this study, we utilized dimethyloxallyl glycine (DMOG), a small-molecule inhibitor of the TET protein, to impede the enzymatic activity of TET and explore subsequent effects on bovine parthenogenetic embryo development. We first detected the expression of the TET family, consisting of TET1, TET2 and TET3, in bovine MII stage oocytes and found that TET3 is more highly expressed than TET1 and TET2. Treatment with 1 mM DMOG increased 5mC levels (30.4% vs 79.8% at the 8-cell stage for satellite I, 25.3% vs 40.6% at the 8-cell stage for α-satellite, 20.5% vs 73.5% at the blastocyst stage for satellite I and 16.6% vs 30.0% at the blastocyst stage for α-satellite) at every bovine parthenogenetic embryo developmental stage. At the same time, DNA methylation level of satellite DNA and pluripotency gene promoters increased significantly. Real-time PCR analysis results indicated that the transcription levels of NANOG and OCT-4 decreased in the DMOG-treated group. Furthermore, TET inhibition negatively affected blastocyst formation, resulting in a decline in the blastocyst rate (17.1 ± 1.3% vs 24.1 ± 0.6%); however, the percentage of apoptotic cells was significantly increased according to the results of a TUNEL assay. Additionally, expression levels of the apoptosis-related gene BAX were up-regulated, while the expression of BCL-2 was down-regulated. In conclusion, these results support that TET plays important roles in bovine parthenogenetic embryo development by influencing DNA methylation reprogramming, gene expression and apoptosis.

5-Formylcytosine Yields DNA-Protein Cross-Links in Nucleosome Core Particles.

In situ generation of 5-formylcytosine (5fC) in nucleosome core particles (NCPs) reveals that 5fC leads to essential DNA-protein cross-links (DPCs). Mechanistic studies using chemical models and mutated histones demonstrate that DPCs form reversibly between the formyl function of 5fC and primary amines on histones. These results suggest that DPC formation from 5fC in chromatin occurs in addition to its role in DNA demethylation.

Differential expression of ten-eleven translocation genes in endometrial cancers.

Ten-eleven translocation proteins are α-ketoglutarate-dependent dioxygenases involved in the conversion of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine, and 5-carboxylcytosine that play a significant role in DNA demethylation. Deregulation of TET genes expression and changes in the level of 5-hmC are thought to be associated with the onset and progression of several types of cancer, but there are no such data related to endometrial cancer. The aim of the work was to investigate the messenger RNA expression levels of TET1, TET2, and TET3 in relation to clinicopathological characteristics of endometrial cancer as well as the correlation between expression of TET genes and the level of 5-hmC/5-mC. The prognostic significance of TETs expression for overall survival was established. We found that TET1 and TET2 messenger RNA expression was lower and TET3 was higher in cancers compared to normal tissues. Positive correlation between 5-hmC and the relative expression of TET1 and TET2 was found, but no correlation was observed in the case of TET3. Decreased expression of TET1 and TET2 was significantly associated with increased lymph node metastasis and International Federation of Gynecology and Obstetrics stage. Kaplan-Meier analysis indicated that low TET1 expression predicted poor overall survival (p = 0.038). Multivariate analysis identified the TET1 expression in endometrial cancer as an independent prognostic factor. Our results suggest that decreased expression of TET1 correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.