TET2 Knockdown Research Articles

TET3-overexpressing macrophages promote endometriosis.

Endometriosis is a debilitating, chronic inflammatory disease affecting ~10% of reproductive age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we reported identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We showed that factors from the disease microenvironment upregulated TET3 expression transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated pro-inflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival, hence vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation both in human and mouse macrophages. This degradation was dependent on a VHL E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.

Alterations in DNA 5-hydroxymethylation patterns in the hippocampus of an experimental model of chronic epilepsy

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.

TET1 inhibits the migration and invasion of cervical cancer cells by regulating autophagy

ABSTRACT Methylation modifications play pertinent roles in regulating gene expression and various biological processes. The silencing of the demethylase enzyme TET1 can affect the expressions of key oncogenes or tumour suppressor genes, thus contributing to tumour formation. Nonetheless, how TET1 affects the progression of cervical cancer is yet to be elucidated. In this study, we found that the expression of TET1 was significantly downregulated in cervical cancer tissues. Functionally, TET1 knockdown in cervical cancer cells can promote cell proliferation, migration, invasion, cervical xenograft tumour formation and EMT. On the contrary, its overexpression can reverse the aforementioned processes. Moreover, the autophagy level of cervical cancer cells can be enhanced after TET1 knockdown. Mechanistically, methylated DNA immunoprecipitation (MeDIP)-sequencing and MeDIP quantitative real-time PCR revealed that TET1 mediates the methylation of autophagy promoter regions. These findings suggest that TET1 affects the autophagy of cervical cancer cells by altering the methylation levels of NKRF or HIST1H2AK, but the specific mechanism needs to be investigated further.

Skeletal muscle TET3 promotes insulin resistance through destabilisation of PGC-1α

Aim/hypothesisThe peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) plays a critical role in the maintenance of glucose, lipid and energy homeostasis by orchestrating metabolic programs in multiple tissues in response to environmental cues. In skeletal muscles, PGC-1α dysregulation has been associated with insulin resistance and type 2 diabetes but the underlying mechanisms have remained elusive. This research aims to understand the role of TET3, a member of the ten-eleven translocation (TET) family dioxygenases, in PGC-1α dysregulation in skeletal muscles in obesity and diabetes.MethodsTET expression levels in skeletal muscles were analysed in humans with or without type 2 diabetes, as well as in mouse models of high-fat diet (HFD)-induced or genetically induced (ob/ob) obesity/diabetes. Muscle-specific Tet3 knockout (mKD) mice were generated to study TET3’s role in muscle insulin sensitivity. Genome-wide expression profiling (RNA-seq) of muscle tissues from wild-type (WT) and mKD mice was performed to mine deeper insights into TET3-mediated regulation of muscle insulin sensitivity. The correlation between PGC-1α and TET3 expression levels was investigated using muscle tissues and in vitro-derived myotubes. PGC-1α phosphorylation and degradation were analysed using in vitro assays.ResultsTET3 expression was elevated in skeletal muscles of humans with type 2 diabetes and in HFD-fed and ob/ob mice compared with healthy controls. mKD mice exhibited enhanced glucose tolerance, insulin sensitivity and resilience to HFD-induced insulin resistance. Pathway analysis of RNA-seq identified ‘Mitochondrial Function’ and ‘PPARα Pathway’ to be among the top biological processes regulated by TET3. We observed higher PGC-1α levels (~25%) in muscles of mKD mice vs WT mice, and lower PGC-1α protein levels (~25–60%) in HFD-fed or ob/ob mice compared with their control counterparts. In human and murine myotubes, increased PGC-1α levels following TET3 knockdown contributed to improved mitochondrial respiration and insulin sensitivity. TET3 formed a complex with PGC-1α and interfered with its phosphorylation, leading to its destabilisation.Conclusions/interpretationOur results demonstrate an essential role for TET3 in the regulation of skeletal muscle insulin sensitivity and suggest that TET3 may be used as a potential therapeutic target for the metabolic syndrome.Data availabilitySequences are available from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with accession number of GSE224042.Graphical

Promoter methylation and increased expression of PD-L1 in patients with active tuberculosis.

The PD-1/PD-L1 pathway plays a pivotal role in T cell activity and is involved in the pathophysiology of Mycobacterium tuberculosis (MTB) infection. DNA methylation is a mechanism that modulates PD-L1 expression in cancer cells. However, its effect on PD-L1 expression in macrophages after MTB infection remains unknown. We prospectively enrolled patients with active tuberculosis (TB) and non-TB subjects. The expression of PD-L1 and methylation-related genes in peripheral blood mononuclear cells (PBMCs) were investigated and their correlation with disease severity and treatment outcomes were examined. PD-L1 promoter methylation status was evaluated using bisulfite sequencing. Immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to visualize PD-L1- and TET-1-expressing cells in lung tissues from patients with TB and in macrophage cell lines with MTB-related stimulation. In total, 80 patients with active TB and 40 non-TB subjects were enrolled in the analysis. Patients with active TB had significantly higher expression of PD-L1, DNMT3b, TET1, TET2, and lower expression of DNMT1, compared to that in the non-TB subjects. The expression of PD-L1 and TET-1 was significantly associated with 1-month smear and culture non-conversion. IHC and IF staining demonstrated the co-localization of PD-L1- and TET-1-expressing macrophages in patients with pulmonary TB and in human macrophage cell lines after MTB-related stimulation. DNMT inhibition and TET-1 knockdown in human macrophages increased and decreased PD-L1 expression, respectively. Overall, PD-L1 expression is increased in patients with active TB and is correlated with treatment outcomes. DNA methylation is involved in modulating PD-L1 expression in human macrophages.

Specific inhibition of TET1 in the spinal dorsal horn alleviates inflammatory pain in mice by regulating synaptic plasticity

DNA demethylation mediated by ten-eleven translocation 1 (TET1) is a critical epigenetic mechanism in which gene expression is regulated via catalysis of 5-methylcytosine to 5-hydroxymethylcytosine. Previously, we demonstrated that TET1 is associated with the genesis of chronic inflammatory pain. However, how TET1 participates in enhanced nociceptive responses in chronic pain remains poorly understood. Here, we report that conditional knockout of Tet1 in dorsal horn neurons via intrathecal injection of rAAV-hSyn-Cre in Tet1fl/fl mice not only reversed the inflammation-induced upregulation of synapse-associated proteins (post-synaptic density protein 95 (PSD95) and synaptophysin (SYP)) in the dorsal horn but also ameliorated abnormalities in dendritic spine morphology and alleviated pain hypersensitivities. Pharmacological blockade of TET1 by intrathecal injection of a TET1-specific inhibitor—Bobcat 339—produced similar results, as did knockdown of Tet1 by intrathecal injection of siRNA. Thus, our data strongly suggest that increased TET1 expression during inflammatory pain upregulates the expression of multiple synapse-associated proteins and dysregulates synaptic morphology in dorsal horn neurons, suggesting that Tet1 may be a potential target for analgesic strategies.

Saikosaponin a activates tet1/dll3/notch1 signalling and promotes hippocampal neurogenesis to improve depression-like behavior in mice

Ethnopharmacological relevanceRadix Bupleuri, also named “Chaihu” in Chinese, is a substance derived from the dry roots of Bupleurum chinense DC. [Apiaceae] and Bupleurum scorzonerifolium Willd. [Apiaceae]. Radix Bupleuri was initially recorded as a medicinal herb in Shen Nong Ben Cao Jing, the earliest monograph concerning traditional Chinese medicine (TCM). Ever since, Radix Bupleuri has been broadly used to alleviate exterior syndrome, disperse heat, modulate the liver-qi, and elevate yang-qi in TCM. Radix Bupleuri has also been utilized as an important component in Xiaoyaosan, a classical formula for relieving depression, which was originated from the famous Chinese medical book called “Tai Ping Hui Min He Ji Ju Fang” in Song Dynasty. Currently, many valuable pharmacological effects of Radix Bupleuri have been explored, such as antidepressant, neuroprotective activities, antiinflammation, anticancer, immunoregulation, etc. Former studies have illustrated that Saikosaponin A (SSa), one of the primary active components of Radix Bupleuri, possesses potential antidepressant properties. However, the underlying mechanisms still remain unknown. Aim of the studyWe used a chronic social defeat stress (CSDS) mouse model to explore the ameliorative effects and potential mechanisms of SSa in depressive disorder in vivo. Materials and methodsThe CSDS mouse model was established and mice underwent behavioral studies using assays such as the social interaction test (SIT), sucrose preference test (SPT), forced-swim test (FST), tail suspension test (TST), and open field test (OFT). Western blotting, immunofluorescence, and Golgi staining were performed to investigate signaling pathway activity, and alterations in synaptic spines in the hippocampus. To model the anticipated interaction between SSa and Tet1, molecular docking and microscale thermophoresis (MST) techniques were employed. Finally, sh-RNA Tet1 was employed for validation via lentiviral transfection in CSDS mice to confirm the requirement of Tet1 for SSA efficacy. ResultsSSa dramatically reduced depressed symptoms, boosted the expression of Tet1, Notch, DLL3, and BDNF, encouraged hippocampus development, and enhanced the dendritic spine density of hippocampal neurons. In contrast, Tet1 knockdown in CSDS mice dampened the beneficial effects of SSa on depressive symptoms. ConclusionsTherefore, our results suggest that SSa significantly activates the Tet1/Notch/DLL3 signaling pathways and promotes hippocampal neurogenesis to exert antidepressant effects in the CSDS mouse model in vivo. The present results also provide new insight into the importance of the Tet1/DLL3/Notch pathways as potential targets for novel antidepressant development.

ZMIZ1 Upregulation of TET3-Mediated Hydroxymethylation Induces M2 Polarization of Kupffer Cells in Hepatocellular Carcinogenesis by Mediating Notch1/c-Myc Signaling

Hydroxymethylation of DNA, mediated by the ten-eleven translocation (TET) family of methylcytosine dioxygenases, represents a crucial epigenetic modification that manipulates gene expression in numerous biological processes. This study focuses on the effect of TET3 on the polarization of Kupffer cells (KCs) and its connection to the development of hepatocellular carcinoma (HCC). TET3 was found to be abundant in KCs, and its knockdown induced an M2–M1 phenotype shift, resulting in the suppression of viability, migration, and invasion of cocultured HCC cells. Additionally, the TET3 knockdown inhibited the tumorigenesis of HCC cells in nude mice. Downstream targets of TET3 were predicted using bioinformatics. TET3-mediated DNA hydroxymethylation of zinc finger MIZ-type containing 1 (ZMIZ1) promoter. The ZMIZ1 protein interacted with notch receptor 1 (Notch1) protein to activate the transcription of c-Myc. Silencing of ZMIZ1 in KCs similarly suppressed M2 polarization of KCs and malignant phenotype of cocultured HCC cells. However, these changes were counteracted by the overexpression of either Notch1 or c-Myc overexpression in KCs. In summary, this study demonstrates that TET3-mediated hydroxymethylation of ZMIZ1 enhances hepatocellular carcinogenesis by promoting M2 skewing of KCs through the Notch1/c-Myc axis.

TET2 inhibits the proliferation and metastasis of lung adenocarcinoma cells via activation of the cGAS-STING signalling pathway

BackgroundEffective identification and development of new molecular methods for the diagnosis, treatment and prognosis of lung adenocarcinoma (LUAD) remains an urgent clinical need. DNA methylation patterns at cytosine bases in the genome are closely related to gene expression, and abnormal DNA methylation is frequently observed in various cancers. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA methylation reversal. This study aimed to explore the role of the TET2 protein and its downstream effector, 5-hmC/5-mC DNA modification, in LUAD progression.MethodsThe expression of TET2 was analysed by real-time PCR, Western blotting and immunohistochemistry. The 5-hmC DNA content was determined by a colorimetric kit. Activation of the cGAS-STING signalling pathway was evaluated by Western blotting. CCK-8, wound healing and Transwell assays were performed to evaluate the effect of TET2 on cell proliferation, migration and invasion abilities. A xenograft model was used to analyse the effect of TET2 on the tumorigenic ability of A549 cells.ResultsTET2 overexpression decreased proliferation and metastasis of A549 and H1975 cells in vitro and in vivo. However, TET2 knockdown dramatically enhanced the proliferation, migration and invasion of A549 and H1975 cells. Mechanistically, activation of the cGAS-STING signalling pathway is critical for the TET2-mediated suppression of LUAD cell tumorigenesis and metastasis.ConclusionIn this study, we demonstrate a tumour suppressor role of TET2 in LUAD, providing new potential molecular therapeutic targets and clinical therapies for patients with non-small cell lung cancer.

MDM2 Antagonist Nutlin-3 Stimulates Global DNA Hydroxymethylation by Enhancing p53-TET1 Signaling Axis.

DNA hydroxymethylation is involved in many biological processes, including nuclear reprogramming, embryonic development, and tumor suppression. In this study, we report that an anticancer agent, nutlin-3, selectively stimulates global DNA hydroxymethylation in TP53 wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine (5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNA hydroxymethylation in TP53-mutated cancer cells. Consistently, nutlin-3 as a MDM2 antagonist only activates wild-type but not mutated TP53. Furthermore, nutlin-3 does not alter the expression of TET1 but slightly reduces the expression of TET2 and TET3 proteins. These TET family proteins are responsible for converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1 knockdown could significantly block the nutlin-3-induced DNA hydroxymethylation as well as TP53 and P21 activation. Immunoprecipitation analysis supports that p53 strongly interacts with TET1 proteins. These results suggest that nutlin-3 activates TP53 and promotes p53-TET1 interaction. As positive feedback, the p53-TET1 interaction further enhances p53 activation and promotes apoptosis. Collectively, we demonstrate that nutlin-3 stimulates DNA hydroxymethylation and apoptosis via a positive feedback mechanism.

Tet2 acts in the lateral habenula to regulate social preference in mice

The lateral habenula (LHb) has been considered a moderator of social behaviors. However, it remains unknown how LHb regulates social interaction. Here, we show that the hydroxymethylase Tet2 is highly expressed in the LHb. Tet2 conditional knockout (cKO) mice exhibit impaired social preference; however, replenishing Tet2 in the LHb rescues social preference impairment in Tet2 cKO mice. Tet2 cKO alters DNA hydroxymethylation (5hmC) modifications in genes that are related to neuronal functions, as is confirmed by miniature two-photon microscopy data. Further, Tet2 knockdown in the glutamatergic neurons of LHb causes impaired social behaviors, but the inhibition of glutamatergic excitability restores social preference. Mechanistically, we identify that Tet2 deficiency reduces 5hmC modifications on the Sh3rf2 promoter and Sh3rf2 mRNA expression. Interestingly, Sh3rf2 overexpression in the LHb rescues social preference in Tet2 cKO mice. Therefore, Tet2 in the LHb may be a potential therapeutic target for social behavior deficit-related disorders such as autism.

Abstract 2226: Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression

Abstract Loss of function in DNA methylation regulators such as the Ten-eleven translocation (TET1-3) proteins, have been implicated in the progression of lymphoma. TET enzymes constitute a family of iron and αKG-dependent dioxygenases that oxidize 5-methylcytosine in DNA to promote demethylation and the reversal of gene silencing. The TET enzymes require vitamin C for their optimal catalytic activity, however, many patients with hematological malignancies are vitamin C deficient. Enhancing and restoring TET function with high-dose vitamin C has been shown to block the progression of solid tumors and blood malignancies. A combination of genetic and epigenetic diversity in lymphoma can result in the aberrant overexpression of oncogenes, loss of tumor suppressor function, and genomic instability. Our research models Bcl2-driven B cell lymphoma with Tet-deficiency using transgenic RNAi for the knockdown of Tet1 or Tet2. In vitro, our results have shown that this model shows increased self-renewal capacity of early B cell progenitors and precursors that can be suppressed by genetic restoration of Tet expression or upon treatment with vitamin C. In vivo, the B cell compartment of mice with Bcl2 overexpression and Tet1 or Tet2 deficiency exhibit a block in B cell maturation during a long period of pre-malignancy, and a decreased frequency of follicular B cells in the spleen from 3 months of age. Our aim is to determine mechanistically the effects of Tet-deficiency on B cell lymphoma progression and whether vitamin C can restore Tet activity to suppress lymphoma progression. Citation Format: Minh Quang Lam, Luisa Cimmino. Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2226.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype

Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Epigenetic Regulation of Corneal Epithelial Differentiation by TET2.

Epigenetic DNA modification by 5-hydroxymethylcytosine (5hmC), generated by the Ten-eleven translocation (TET) dioxygenases, regulates diverse biological functions in many organ tissues, including the mammalian eye. For example, 5hmC has been shown to be involved in epigenetic regulation of retinal gene expression. However, a functional role of 5hmC in corneal differentiation has not been investigated to date. Here, we examined 5hmC and TET function in the human cornea. We found 5hmC highly expressed in MUC16-positive terminally differentiated cells that also co-expressed the 5hmC-generating enzyme TET2. TET2 knockdown (KD) in cultured corneal epithelial cells led to significant reductions of 5hmC peak distributions and resulted in transcriptional repression of molecular pathways involved in corneal differentiation, as evidenced by downregulation of MUC4, MUC16, and Keratin 12. Additionally, integrated TET2 KD RNA-seq and genome-wide Reduced Representation Hydroxymethylation Profiling revealed novel epigenetically regulated genes expressed by terminally differentiated cells, including KRT78, MYEOV, and MAL. In aggregate, our findings reveal a novel function of TET2 in the epigenetic regulation of corneal epithelial gene expression and identify novel TET2-controlled genes expressed in differentiated corneal epithelial cells. These results point to potential roles for TET2 induction strategies to enhance treatment of corneal diseases associated with abnormal epithelial maturation.

Loss of TET2 impairs endothelial angiogenesis via downregulating STAT3 target genes

BackgroundIschemic diseases represent a major global health care burden. Angiogenesis is critical in recovery of blood flow and repair of injured tissue in ischemic diseases. Ten–eleven translocation protein 2 (TET2), a member of DNA demethylases, is involved in many pathological processes. However, the role of TET2 in angiogenesis is still unrevealed.MethodsTET2 was screened out from three DNA demethylases involved in 5-hydroxylmethylcytosine (5-hmC) regulation, including TET1, TET2 and TET3. Knockdown by small interfering RNAs and overexpression by adenovirus were used to evaluate the role of TET2 on the function of endothelial cells. The blood flow recovery and density of capillary were analyzed in the endothelial cells-specific TET2-deficient mice. RNA sequencing was used to identify the TET2-mediated mechanisms under hypoxia. Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation-qPCR (ChIP-qPCR) and glucosylated hydroxymethyl-sensitive-qPCR (GluMS-qPCR) were further performed to reveal the interaction of TET2 and STAT3.ResultsTET2 was significantly downregulated in endothelial cells under hypoxia and led to a global decrease of 5-hmC level. TET2 knockdown aggravated the hypoxia‐induced dysfunction of endothelial cells, while TET2 overexpression alleviated the hypoxia‐induced dysfunction. Meanwhile, the deficiency of TET2 in endothelial cells impaired blood flow recovery and the density of capillary in the mouse model of hindlimb ischemia. Mechanistically, RNA sequencing indicated that the STAT3 signaling pathway was significantly inhibited by TET2 knockdown. Additionally, Co-IP, ChIP-qPCR and GluMS-qPCR further illustrated that STAT3 recruited and physically interacted with TET2 to activate STAT3 target genes. As expected, the effects of TET2 overexpression were completely suppressed by STAT3 silencing in vitro.ConclusionsOur study suggests that the deficiency of TET2 in endothelial cells impairs angiogenesis via suppression of the STAT3 signaling pathway. These findings give solid evidence for TET2 to be a therapeutic alternative for ischemic diseases.Graphical

TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway.

Ten-eleven translocation 3 (TET3) participates in tumorigenesis and malignant transformation by mediating DNA demethylation and specific gene activation in malignances. This study aims to elucidate its molecular function and regulatory mechanism in esophageal squamous cell carcinoma (ESCC). Stable ESCC cells that infected with TET3 overexpression (OE) and knockdown lentiviral vector had been established. The biological behaviors and molecular mechanism of TET3 were demonstrated by cell biology experiments in vitro and in vivo. Tissues from patients with ESCC were used to demonstrate the clinical value of TET3. Our findings revealed that TET3 is highly expressed in ESCC tissues and related to poor prognosis of patients with ESCC. OE of TET3 presented a significant effect on proliferation, metastatic potential, and spheroid formation of ESCC cells by activating the PI3K/AKT/GSK3β/β-catenin axis. Knockdown of TET3 could remarkably reverse these malignant phenotypes. Patients with ESCC with high TET3 expression resulted in a shorter overall survival (OS) and disease-free survival. Based on the multivariate analysis, TET3 could be an independent favorable factor for predicting OS and recurrence. The high expression of TET3 not only aggravates malignant behaviors in vitro and in vivo but also becomes a novel biomarker for clinical monitoring and individualized precision treatment for patients with ESCC.

Oxidative low-density lipoprotein oxLDL induces pyroptosis in trophoblast cells HTR-8/Svneo by downregulating TET2

ObjectiveTo investigate pyroptosis in the trophoblast cells HTR-8/Svneo induced by TET2 in oxidized low-density lipoprotein (oxLDL). MethodsHTR-8/Svneo cells were treated with vehicle (0 mg/L) and 50 mg/L oxLDL for 24 h. The effects of oxLDL on TET2 expression in HTR-8/Svneo cells were detected by Western blot. Knockdown of TET2 was performed in HTR-8/Svneo cells using si-TET2. CCK8, Transwell, and wound healing assays were used to detect the effect of TET2 knockdown on the proliferation, cell cycle, migration, and invasion of HTR-8/Svneo cells, respectively. Western blot was used to quantify the expression levels of the pyroptosis proteins GSDMD and caspase-1 in si-TET2 treated cells. ResultsoxLDL downregulates TET2 expression in a concentration-dependent manner. TET2 knockdown inhibits HTR-8/Svneo invasion and migration but promotes cell proliferation. Western blot results showed that TET2 knockdown upgregulated the expression of GSDMD and caspase-1. ConclusionoxLDL induces pyroptosis of HTR-8/Svneo by downregulating TET2 expression, resulting in decreased trophoblast invasion and migration. These data suggest that TET2-induced pyroptosis play a critical role in gestational pathologies, such as preeclampsia. Further studies can clarify the mechanisms of this process and elucidate potential preventive and therapeutic targets.

Demonstration That Loss of TET2 Impairs DNA Mismatch Repair Resulting in Clonally Restricted Genomic Instability

Epigenetic dysregulation is a common feature of myeloid neoplasia (MN). TET2, along with TET1 and TET3 are the only known enzymes, which progressively oxidize 5-methyl cytosine and thus facilitate active demethylation of CpG islands, a process critical for efficient gene expression and maintenance of genomic integrity. Loss of function TET2 mutations (TET2MT) are one of the most common somatic lesions in MN. To date, the precise mechanisms of TET2MT in leukemogenesis remain unclear. However, two potential mechanisms have been postulated; i) TET2MT may lead to expansion of stem cells by differentiation block and/or increased self-renewal ii) clonally restricted hyper mutations resulting in acquisition of secondary driver mutations. This functional duality may explain proleukemogenic consequences of TET2MT. Both mechanisms are compatible with their relatively moderate driver phenotype. Increased CàT transitions via deamination of 5mC in TET2MT has been postulated; however this report focuses on a novel mechanism of TET2MT- mediated impairment of mismatch repair (MMR). WES was used for analysis of the mutational status of 435 MN cases to test whether TET2MT (n= 82/435; 19%) predispose to acquisition of additional mutations. Following exclusion of dysfunction in known DNA repair genes, we compared the median SNVs in TET2MT vs. TET2WT. TET2MT cases had nearly a 2-fold increased median SNVs (p<0.0001) vs. TET2WT including a 2.1-fold increase in truncations (p=0.03) with a linear correlation (r=0.7) of SNVs vs. clonality, consistent with a gene-dose effect. ShRNA knockdown of TET2 in HeLa and CRISPR knockout in HEK293 cells resulted in a 24- and 30-fold increase in spontaneous mutations in the HPRT assay. The heightened spontaneous mutability in TET2KO/KD cells were reversed by ectopic expression of TET2. In Tet2-/- mice, ~1.5-fold increased mutagenicity at TET2-dependent active demethylation sites were detected suggesting its direct or indirect involvement in one or more DNA repair pathways. Consistent with our hypothesis, endogenous co-immunoprecipitation of TET2 followed by tandem-affinity purification and characterization of protein complexes with LCMS/MS in 4 different systems including human leukemic cell lines (K562 and CMK), murine embryonic stem cell and erythroid leukemia cells MEL identified all the major components of DNA MMR complex as a part of TET2 interactome. In every cell line, MSH6, a main component of MMR complex was an abundant TET2 interacting protein. This result was further confirmed by reverse co-immunoprecipitation using anti-MSH6 antibodies in different cell lines. Using truncating deletion constructs of TET2, MSH6 binding site was mapped on TET2 N-terminal amino acid segment 460-942. Functional MMR fluorescence reporter assay, wherein a point mutant of GFP, if repaired, is converted to BFP, demonstrated that loss of TET2 indeed compromised the rate of MMR in CRISPR generated deletion of TET2 in K562 and HEK293 cells. Ectopic expression of N-terminal (aa 460-942) MSH6 binding domain rescued this defect. The genomic instability due to MMR dysfunction in TET2MT cells may allow therapeutic targeting of DNA repair proteins in a subset of TET2MT MN. Therefore, we tested the therapeutic vulnerability of TET2MT cells against small molecule DNA repair inhibitors (e.g., clinically approved PARP antagonists) in engineered isogenic and natural TET2MT cell lines. The TET2KO/KD cells were 2-10X more sensitive to olaparib and rukaparib compared to parental TET2 proficient cells, both under steady state and under conditions of oxidative stress as observed in gH2AX assays and various apoptotic readouts. In summary, for the first time here we report that TET2 directly binds to MSH6 and recruits MMR complex to the site of mismatch around CpG islands and facilitate the repair. Loss of TET2impairs this process, explaining the accumulation of secondary hits in mutant clones, which by this virtue, progressively increases their leukemogenic fitness. Given that MSH6 is known to interact 5hmC, TET2 may guide MMR to TET2-dependent DNA loci directly linking TET2MTto genomic instability a biologic feature that can be therapeutically exploited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interactive regulation of DNA demethylase gene TET1 and m6A methyltransferase gene METTL3 in myoblast differentiation

DNA methylation (5mC) and mRNA N6-methyladenosine (m6A) play an essential role in gene transcriptional regulation. DNA methylation has been well established to be involved in skeletal muscle development. Interacting regulatory mechanisms between DNA methylation and mRNA m6A modification have been identified in a variety of biological processes. However, the effect of m6A on skeletal muscle differentiation and the underlying mechanisms are still unclear. It is also unknown whether there is an interaction between DNA methylation and mRNA m6A modification in skeletal myogenesis. In the present study, we used m6A-IP-qPCR, LC–MS/MS and dot blot assays to determine that the DNA demethylase gene, TET1, exhibited increased m6A levels and decreased mRNA expression during bovine skeletal myoblast differentiation. Dual-luciferase reporter assays and RIP experiments demonstrated that METTL3 suppressed TET1 expression by regulating TET1 mRNA stability in a m6A-YTHDF2-dependent manner. Furthermore, TET1 mediated DNA demethylation of itself, MYOD1 and MYOG, thereby stimulating their expression to promote myogenic differentiation. Ectopic expression of TET1 rescued the effect of METTL3 knockdown on reduced myotubes. In contrast, TET1 knockdown impaired the myogenic differentiation promoted by METTL3 overexpression. Moreover, ChIP experiments found that TET1 could bind and demethylate METTL3 DNA, which enhanced METTL3 expression. In addition, TET1 knockdown decreased m6A levels. ChIP assays also showed that TET1 knockdown contributed to the binding of H3K4me3 and H3K27me3 to METTL3 DNA. Our results revealed a negative feedback regulatory loop between TET1 and METTL3 in myoblast differentiation, which unveiled the interplay among DNA methylation, RNA methylation and histone methylation in skeletal myogenesis.

Knockdown of Tet2 Inhibits the Myogenic Differentiation of Chicken Myoblasts Induced by Ascorbic Acid.

Ascorbic acid (also called Vitamin C, VC) strengthens the function of Tets families and directly increases DNA demethylation level to affect myogenic differentiation. However, the precise regulatory mechanism of DNA methylation in chicken myogenesis remains unclear. Results of present study showed that the mRNA expression of MyoD significantly decreased and MyoG and MyHC increased in myoblasts treated with 5 μM 5-azacytidine (5-AZA) and 5 μM VC (p &lt; 0.05). Results also indicated the formation of myotubes was induced by 5-AZA or VC, but this effect was attenuated after knockdown of Tet2. In addition, the protein expression of TET2, DESMIN and MyHC was remarkable increased by the addition of 5-AZA or VC, and the upregulation was inhibited after knockdown of Tet2 (p &lt; 0.05). DNA dot blot and immunofluorescence staining results suggested that the level of 5hmC was significantly increased when treated with 5-AZA or VC, even by Tet2 knockdown (p &lt; 0.05). Moreover, 5-AZA and VC reduced the level of dimethylation of lysine 9 (H3K9me2) and trimethylation of lysine 27 of histone 3 (H3K27me3), and this inhibitory effect was eliminated after Tet2 knockdown (p &lt; 0.05). These data indicated that Tet2 knockdown antagonized the increased levels of 5hmC and H3K27me3 induced by 5-AZA and VC, and eventually reduced myotube formation by modulating the expression of genes involved in myogenic differentiation. This study provides insights that epigenetic regulators play essential roles in mediating the myogenic program of chicken myoblasts.