Ten-eleven Translocation Protein 1 Research Articles

Epigenetic regulation of DNA repair gene program by Hippo/YAP1-TET1 axis mediates sorafenib resistance in HCC

Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.Graphical

FMRP promotes transcription-coupled homologous recombination via facilitating TET1-mediated m5C RNA modification demethylation

RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, pro-moting transcription-coupled homologous recombination (HR). Here, we identifiedthat Fragile X mental retardation protein (FMRP) promotes transcription-coupled HRvia its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleventranslocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporalorder at the transcriptionally active sites of DSBs. FMRP displays a higher affinity forDNA:RNA hybrids containing m5C-modified RNA than for hybrids without modifica-tion and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chroma-tin- or RNA-binding domain of FMRP compromises demethylation of damage-inducedm5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unre-solved R-loop and m5C preventing completion of DSB repair, FMRP depletion or lowexpression leads to delayed repair of DSBs at transcriptionally active sites and sensitizescancer cells to radiation in a BRCA-independent manner. Together, ourfindings presentan m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraserto promote mRNA-dependent repair and cell survival in cancer.

Assay for TET1 activity and its inhibitors screening with signal amplification by both nanoparticles and Ru(III) redox recycling

Ten-eleven translocation protein 1 (TET1) is one member of TET proteins family which plays a key role in dynamic DNA methylation-demethylation process. Herein, a novel biosensor was constructed for TET1 detection and its inhibitors screening utilizing restriction digestion of endonuclease enzyme MspI. Half-methylated oligonucleotide (5mC DNA) was used as target and Ru(NH3)63+ as electrochemical signal probe. After the treatment by TET1 and T4 β-glucosyltransferase (T4 β-GT), target oligonucleotide would not be recognized and digested. If there was no TET1, the target would be digested and the response of biosensor decreased greatly. The current difference of biosensor with and without the incubation with TET1 was therefore dependent on the concentration of TET1. To increase sensitivity of the biosensor, nanostructured film at electrode surface and nanoparticles modified oligonucleotides were employed as signal amplification elements for Ru(NH3)63+ recycling. Finally, this biosensor showed high performance with a wide linear range of TET1 concentration from 3.5–21 ng/μL and a low detection limit of 0.33 ng/μL, which is superior to other existing methods. The inhibition effects of Bobcat339 on TET1 was successfully proved by our biosensor with an IC50 of 38 μM. Not only that, but the feasibility of the biosensor for inhibitors screening was evaluated and further confirmed by other compounds including two anticancer drugs and three active ingredients of traditional Chinese medicine.

Identification of DNA (de)methylation-related genes and their transcriptional response to environmental challenges in an invasive model ascidian.

Marine invasive species are constantly challenged by acute or recurring environmental stresses during their range expansions. DNA methylation-mediated stress memory has been proposed to effectively affect species' response and enhance their overall performance in recurring environmental challenges. In order to further test this proposal in marine invasive species, we identified genes in the DNA methylation and demethylation processes in the highly invasive model species, Ciona robusta, and subsequently investigated the expression patterns of these genes under recurring salinity stresses. After a genome-wide comprehensive survey, we found a total of six genes, including two genes of DNA methyltransferase 3a (DNMT3a1 and DNMT3a2), and one gene of DNA methyltransferase 1 (DNMT1), methyl-CpG-binding domain protein 2 (MBD2), methyl-CpG-binding domain protein 4 (MBD4) and ten-eleven-translocation protein 1 (TET1). Phylogenetic reconstruction and domain arrangement analyses showed that the deduced proteins of the identified genes were evolutionarily conserved and functionally similar with their orthologs. All genes were constitutively expressed in all four tested tissues. Interestingly, we found time-dependent and stress-specific gene expression patterns under high and low salinity stresses. Under the recurring high salinity stresses, DNMT3a1 and TET1 conformed to the definition of memory genes, while under the recurring low salinity stresses, two DNMT3a paralogues were identified as the memory genes. Altogether, our results clearly showed that the transcriptional patterns of (de)methylation-related genes were significantly influenced by environmental stresses, and the transcriptional memory of some (de)methylation-related genes should play crucial roles in DNA methylation-mediated stress memory during the process of biological invasions.

Novel label-free electrochemical strategy for sensitive determination of ten-eleven translocation protein 1

A label-free electrochemical method was developed for sensitive determination of ten-eleven translocation protein 1 (TET1) which can mediate the demethylation of DNA. This strategy is mainly based on MspI-mediated restriction endonuclease reaction. Current response difference of the biosensor before and after cleavage by MspI was dependent on the activity and concentration of TET1. With the aid of Au nanoparticles, this method shows a good linear range from 0.0042 μg μL−1 to 0.0210 μg μL −1 with a correlation coefficient of 0.9350 and a low limit of detection 0.00098 μg μL −1. Finally, this method was used to investigate the effects of n-oxalylglycine (NOG) and taxol on activity of TET1. The results indicated that NOG could inhibit TET1 activity but taxol could not. So this electrochemical biosensor could be applied to TET activity evaluation and inhibitor screening in field of biomedicine and clinical diagnosis.

Effects of maternal feed intake restriction during pregnancy on the expression of growth regulation, imprinting and epigenetic transcription-related genes in foetal goats

Maternal nutrition during gestation is a leading factor of modifying the foetal epigenome and phenotype for mammals. Imprinting genes have important roles in regulating foetal growth, programming and development. There, however, are limited data available on the effects of feed intake restriction on the expression of imprinting genes in pregnant goats. The present study, therefore, was conducted to assess the effects of maternal feed intake restriction on the relative abundance of mRNA for growth imprinting, DNA methyltransferase (DNMT) and epigenetic transcription-related genes in the liver and heart of foetal goats during gestation. A total of 24 Liuyang black goats (2.0±0.3 yr) with similar body weight (BW, 31.22±8.09 kg) and parity (2) were allocated equally to either a control group (CG) or a restriction group (RG) during both early (from 26 to 65 days) and late (from 96 to 135 days) gestation. All goats were fed a mixed diet and had free access to fresh water. The feed of the RG was 40% less than that of the CG. The early and late gestation goats were weighed, bled and slaughtered on days 65 and 135 of gestation, respectively. In early gestation, the foetal weight, body length, the weight of foetal heart and liver were greater (P < 0.05) in the RG. The CpG methylation of genomic DNA in the foetal heart was less (P = 0.0001) in the RG. The relative abundance of mRNA of methyl-CpG-binding domain protein 2 (MBD2) and methyl-CpG-binding domain protein 3 (MBD3) genes in the foetal liver were greater (P < 0.05) in the RG. During the late gestation, the foetal weight, heart weight and liver weight were less (P < 0.05) in the RG. The relative abundance of mRNA for the MBD2 gene (P = 0.043) in the foetal heart, and the ten-eleven translocation protein 1 (TET1) gene (P < 0.05) in both the foetal heart and liver were greater in the RG. These results indicate feed intake restriction during gestation influenced foetal development and regulated the relative abundance of mRNA for epigenetic transcription-related genes.

Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells From Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation.

Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

Hinokitiol induces DNA demethylation via DNMT1 and UHRF1 inhibition in colon cancer cells

BackgroundDNA hypermethylation is a key epigenetic mechanism for the silencing of many genes in cancer. Hinokitiol, a tropolone-related natural compound, is known to induce apoptosis and cell cycle arrest and has anti-inflammatory and anti-tumor activities. However, the relationship between hinokitiol and DNA methylation is not clear. The aim of our study was to explore whether hinokitiol has an inhibitory ability on the DNA methylation in colon cancer cells.ResultsMTT data showed that hinokitiol had higher sensitivity in colon cancer cells, HCT-116 and SW480, than in normal colon cells, CCD18Co. Hinokitiol reduced DNA methyltransferase 1 (DNMT1) and ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression in HCT-116 cells. In addition, the expression of ten-eleven translocation protein 1 (TET1), a known DNA demethylation initiator, was increased by hinokitiol treatment. ELISA and FACS data showed that hinokitiol increased the 5-hydroxymethylcytosine (5hmC) level in the both colon cancer cells, but 5-methylcytosine (5mC) level was not changed. Furthermore, hinokitiol significantly restored mRNA expression of O6-methylguanine DNA methyltransferase (MGMT), carbohydrate sulfotransferase 10 (CHST10), and B-cell translocation gene 4 (BTG4) concomitant with reduction of methylation status in HCT-116 cells.ConclusionsThese results indicate that hinokitiol may exert DNA demethylation by inhibiting the expression of DNMT1 and UHRF1 in colon cancer cells.

Tet1 in Nucleus Accumbens Opposes Depression- and Anxiety-Like Behaviors.

Depression is a leading cause of disease burden, yet current therapies fully treat <50% of affected individuals. Increasing evidence implicates epigenetic mechanisms in depression and antidepressant action. Here we examined a possible role for the DNA dioxygenase, ten-eleven translocation protein 1 (TET1), in depression-related behavioral abnormalities. We applied chronic social defeat stress, an ethologically validated mouse model of depression-like behaviors, and examined Tet1 expression changes in nucleus accumbens (NAc), a key brain reward region. We show decreased Tet1 expression in NAc in stress-susceptible mice only. Surprisingly, selective knockout of Tet1 in NAc neurons of adult mice produced antidepressant-like effects in several behavioral assays. To identify Tet1 targets that mediate these actions, we performed RNAseq on NAc after conditional deletion of Tet1 and found that immune-related genes are the most highly dysregulated. Moreover, many of these genes are also upregulated in the NAc of resilient mice after chronic social defeat stress. These findings reveal a novel role for TET1, an enzyme important for DNA hydroxymethylation, in the brain's reward circuitry in modulating stress responses in mice. We also identify a subset of genes that are regulated by TET1 in this circuitry. These findings provide new insight into the pathophysiology of depression, which can aid in future antidepressant drug discovery efforts.

Abstract 1128: TET1-mediated epigenetic reprogramming switches metabolism and promotes malignant phenotypes of ovarian cancer

Abstract Epigenetic dysregulation is one of the mechanisms involved in ovarian cancer carcinogenesis. Recently identified a new epigenetic modulator ten-eleven translocation protein 1 (TET1), a DNA dioxygenase which is believed in DNA demethylation through a 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) conversion, plays an important role in regulating self-renewal and specification in embryonic stem cells. In addition, it works as a tumor suppressor gene by inhibition of cell invasion, migration and tumor growth in breast and prostate cancer. However, the role of TET1 in ovarian cancer remains unknown. Thus we examed the expression level of TET1 in ovarian cancer tissues. We found that TET1 expression level correlated with cancer staging (p = 0.03) in 88 ovarian cancer from our biobank and poor survival (p = 0.012) in TCGA database. High expression of TET1 was observed in advanced stage, high-grade primary tumor specimens in comparison with normal ovarian surface epithelium (OSE) brushings (p = 0.0005) by NCBI database (GSE18520). These results suggested that TET1 may play some roles in ovarian cancer development, which is different from those previously published in other cancers. To study the function of TET1 in ovarian cancer, we generated TET1 over-expressing and knockdown cell lines model, the expression level and enzymatic activities of TET1 were confirmed by real time PCR, western and 5hmC stain. Here we found that TET1 increases the abilities of cell migration, anchorage-independent growth and promotes tumor growth. In addition, TET1 actives cancer stem markers and enhances the abilities of spheroid formation. Ovarian cancer stem cells (OCSCs) from cell line express high level of TET1 while the differentiated progenies suppress TET1 expression. Moreover, seven of eight patient-derived OCSCs revealed high expressing of TET1 in comparison with its parental cancer cells by quantitative PCR. To further examine the TET1 regulation network, we combined the expression array and MethCap-seq to analyze the epigomic changes. We found that a cluster of target genes which were up-regulated through DNA demethylation were enzymes responsible for oxidative phosphorylation. We investigated metabolic status by Extracellular Flux Analyzer (seahourse) on the TET1-overexpressing cells. Compared with control cells, TET1-overexpressing cells revealed 1.7 fold (p = 0.011) increase of oxygen consumption rate (OCR); while the extracellular acidification rate (ECAR) showed no difference (p = 0.856). This bioenergetic metabolism shift may be due to demethylation of subunits of mitochondria complexs. Taken together, TET1 reprograms the epigenome, shifts the metabolism, increases the malignant phenotypes and confers a poor prognosis of ovarian cancer. Targeting mitochondria on TET1-expressing ovarian cancer patients may provide a new way of personalized therapy. Citation Format: Lin-yu Chen, Rui-Lan Huang, Pearlly S. Yan, Tien-Shuo Huang, Yu-Ping Liao, Jian-Liang Chou, Jora M.J. Lin, Tai-Kuang Chao, Michael W.Y. Chan, Wun-Shaing Wayne Chang, Hung-Cheng Lai. TET1-mediated epigenetic reprogramming switches metabolism and promotes malignant phenotypes of ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1128. doi:10.1158/1538-7445.AM2015-1128

Role of Tet1 and 5-hydroxymethylcytosine in cocaine action.

Ten-eleven translocation (TET) enzymes mediate the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is enriched in brain, and its ultimate DNA demethylation. However, the influence of TET and 5hmC on gene transcription in brain remains elusive. We found that ten-eleven translocation protein 1 (TET1) was downregulated in mouse nucleus accumbens (NAc), a key brain reward structure, by repeated cocaine administration, which enhanced behavioral responses to cocaine. We then identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. Together, these reveal a previously unknown epigenetic mechanism of cocaine action and provide new insight into how 5hmC regulates transcription in brain in vivo.