Tet1-mediated 5hmC regulates hippocampal neuroinflammation via wnt signaling as a novel mechanism in obstructive sleep apnoea leads to cognitive deficit.

BACKGROUND: Obstructive sleep apnoea (OSA) is a sleep-disordered breathing characterized by intermittent hypoxia (IH) that may cause cognitive dysfunction. However, the impact of IH on molecular processes involved in cognitive function remains unclear. METHODS: C57BL / 6 J mice were exposed to either normoxia (control) or IH for 6 weeks. DNA hydroxymethylation was quantified by hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing. ten-eleven translocation 1 (Tet1) was knocked down by lentivirus. Specifically, cognitive function was assessed by behavioral experiments, pathological features were assessed by HE staining, the hippocampal DNA hydroxymethylation was examined by DNA dot blot and immunohistochemical staining, while the Wnt signaling pathway and its downstream effects were studied using qRT-PCR, immunofluorescence staining, and Luminex liquid suspension chip analysis. RESULTS: IH mice showed pathological changes and cognitive dysfunction in the hippocampus. Compared with the control group, IH mice exhibited global DNA hydroxylmethylation in the hippocampus, and the expression of three hydroxylmethylases increased significantly. The Wnt signaling pathway was activated, and the mRNA and 5hmC levels of Wnt3a, Ccnd2, and Prickle2 were significantly up-regulated. Further caused downstream neurogenesis abnormalities and neuroinflammatory activation, manifested as increased expression of IBA1 (a marker of microglia), GFAP (a marker of astrocytes), and DCX (a marker of immature neurons), as well as a range of inflammatory cytokines (e.g. TNFa, IL3, IL9, and IL17A). After Tet1 knocked down, the above indicators return to normal. CONCLUSION: Activation of Wnt signaling pathway by hippocampal Tet1 is associated with cognitive dysfunction induced by IH.

TET1 displays catalytic and non-catalytic functions in the adult mouse cortex.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms.

DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.

ZBTB20 suppresses tumor growth in glioblastoma through activating the TET1/FAS/caspase­3 pathway.

Zinc finger and BTB domain containing 20 (ZBTB20) is a key transcription repressor that regulates multiple physiological and pathophysiological processes. Thus far, the role of ZBTB20 in glioblastoma (GBM), a World Health Organization grade IV glioma, remains unclear. In the present study, the expression profile data of ZBTB20 in GBM tissues from public databases was analyzed. It was found that ZBTB20 expression in GBM tissues was significantly lower than that measured in lower grade glioma tissues. Furthermore, patients with GBM with lower ZBTB20 expression were associated with a shorter overall survival time. Gain- and loss-of-function experiments in GBM cells were also performed. The results demonstrated that ZBTB20 overexpression decreased GBM cell proliferation, while ZBTB20 knockdown significantly enhanced it. Cell cycle analysis showed the ZBTB20 overexpression may have inhibited proliferation through cell cycle arrest at the G2/M phase, while ZBTB20 knockdown increased the percentages of cells in both the S phase and G2/M phase. Ten-eleven translocation 1 (TET1) is an important tumor suppressor involved in the formation of various types of tumor, and it was upregulated in ZBTB20-overexpressing GBM cells. It was further demonstrated that ZBTB20 activated the TET1/FAS/caspase-3 pathway. The results of the present study therefore indicated the potential role of ZBTB20 as a tumor suppressor and therapeutic target for GBM.

Active DNA Demethylase, TET1, Increases Oxidative Phosphorylation and Sensitizes Ovarian Cancer Stem Cells to Mitochondrial Complex I Inhibitor.

Ten-eleven translocation 1 (TET1) is a methylcytosine dioxygenase involved in active DNA demethylation. In our previous study, we demonstrated that TET1 reprogrammed the ovarian cancer epigenome, increased stem properties, and activated various regulatory networks, including metabolic networks. However, the role of TET1 in cancer metabolism remains poorly understood. Herein, we uncovered a demethylated metabolic gene network, especially oxidative phosphorylation (OXPHOS). Contrary to the concept of the Warburg effect in cancer cells, TET1 increased energy production mainly using OXPHOS rather than using glycolysis. Notably, TET1 increased the mitochondrial mass and DNA copy number. TET1 also activated mitochondrial biogenesis genes and adenosine triphosphate production. However, the reactive oxygen species levels were surprisingly decreased. In addition, TET1 increased the basal and maximal respiratory capacities. In an analysis of tricarboxylic acid cycle metabolites, TET1 increased the levels of α-ketoglutarate, which is a coenzyme of TET1 dioxygenase and may provide a positive feedback loop to modify the epigenomic landscape. TET1 also increased the mitochondrial complex I activity. Moreover, the mitochondrial complex I inhibitor, which had synergistic effects with the casein kinase 2 inhibitor, affected ovarian cancer growth. Altogether, TET1-reprogrammed ovarian cancer stem cells shifted the energy source to OXPHOS, which suggested that metabolic intervention might be a novel strategy for ovarian cancer treatment.

TET1-Lipid Nanoparticle Encapsulating Morphine for Specific Targeting of Peripheral Nerve for Pain Alleviation.

Background: Opioids are irreplaceable analgesics owing to the lack of alternative analgesics that offer opioid-like pain relief. However, opioids have many undesirable central side effects. Restricting opioids to peripheral opioid receptors could reduce those effects while maintaining analgesia. Methods: To achieve this goal, we developed Tet1-LNP (morphine), a neural-targeting lipid nanoparticle encapsulating morphine that could specifically activate the peripheral opioid receptor in the dorsal root ganglion (DRG) and significantly reduce the side effects caused by the activation of opioid receptors in the brain. Tet1-LNP (morphine) were successfully prepared using the thin-film hydration method. In vitro, Tet1-LNP (morphine) uptake was assessed in differentiated neuron-like PC-12 cells and dorsal root ganglion (DRG) primary cells. The uptake of Tet1-LNP (morphine) in the DRGs and the brain was assessed in vivo. Von Frey filament and Hargreaves tests were used to assess the antinociception of Tet1-LNP (morphine) in the chronic constriction injury (CCI) neuropathic pain model. Morphine concentration in blood and brain were evaluated using ELISA. Results: Tet1-LNP (morphine) had an average size of 131 nm. Tet1-LNP (morphine) showed high cellular uptake and targeted DRG in vitro. CCI mice treated with Tet1-LNP (morphine) experienced prolonged analgesia for nearly 32 h compared with 3 h with free morphine (p < 0.0001). Notably, the brain morphine concentration in the Tet1-LNP (morphine) group was eight-fold lower than that in the morphine group (p < 0.0001). Conclusion: Our study presents a targeted lipid nanoparticle system for peripheral neural delivery of morphine. We anticipate Tet1-LNP (morphine) will offer a safe formulation for chronic neuropathic pain treatment, and promise further development for clinical applications.

Targeted Demethylation of FOXP3-TSDR Enhances the Suppressive Capacity of STAT6-deficient Inducible T Regulatory Cells.

In vitro induced T regulatory cells (iTregs) are promising for addressing inflammation-driven diseases. However, current protocols for the generation and expansion of iTregs fail to induce extensive demethylation of the Treg-specific demethylated region (TSDR) within the FOXP3 gene, recognized as the master regulator for regulatory T cells (Tregs). This deficiency results in the rapid loss of Foxp3 expression and an unstable regulatory phenotype. Nevertheless, inhibition of STAT6 signaling effectively stabilizes Foxp3 expression in iTregs. Thus, this study aimed to develop a protocol combining epigenetic editing with STAT6 deficiency to improve iTregs' ability to maintain stable suppressive function and a functional phenotype. Our findings demonstrate that the combination of STAT6 deficiency (STAT6-/-) with targeted demethylation of the TSDR using a CRISPR-TET1 tool leads to extensive demethylation of FOXP3-TSDR. Demethylation in STAT6-/- iTregs was associated with enhanced expression of Foxp3 and suppressive markers such as CTLA-4, PD-1, IL-10, and TGF-ß. Furthermore, the edited STAT6-/- iTregs exhibited an increased capacity to suppress CD8+ and CD4+ lymphocytes and could more efficiently impair Th1-signature gene expression compared to conventional iTregs. In conclusion, the deactivation of STAT6 and TSDR-targeted demethylation via CRISPR-TET1 is sufficient to induce iTregs with heightened stability and increased suppressive capacity, offering potential applications against inflammatory and autoimmune diseases.

Reactivation of methylation-silenced PAX1 inhibits cervical cancer proliferation and migration via the WNT/TIMELESS pathway.

Although aberrant methylation of PAX1 is closely associated with cervical cancer (CC), PAX1 methylation (PAX1m) and its role in CC remain to be elucidated. Here, we clarified the biological function of PAX1 in CC. First, PAX1m in ThinPrep cytologic test samples was measured via quantitative methylation-specific PCR. The results showed that PAX1 promoter methylation levels were significantly increased in CC patients (p < 0.001). We also found that PAX1 promoter methylation levels were positively correlated with tumor purity but negatively correlated with immune-infiltration via public databases. Then, CRISPR-based methylation perturbation tools (dCas9-Tet1) were constructed to further demonstrate that DNA methylation participates in the regulation of PAX1 expression directly. Gain- and loss-of-function experiments were used to show that PAX1 overexpression restrained proliferation, migration and improved cisplatin sensitivity by interfering with the WNT/TIMELESS axis in CC cells. Additionally, Co-immunoprecipitation assays further confirmed the interaction between PAX1 and TCF7L2. Taken together, our results suggested that a tumor suppressor role of PAX1 in CC and that CRISPR-based PAX1 demethylation editing might be a promising therapeutic strategy for CC.

TET1 mediated m5C modification of RelB aggravates cerebral ischemia/reperfusion-induced neuroinflammation through regulating microglia polarization.

Microglia mediated neuroinflammation is one of the major contributors to brain damage in cerebral ischemia reperfusion injury (CI/RI). Recently, RNA modification was found to contribute to the regulation of microglia polarization and the subsequent development of cerebral I/R neuroinflammation. Herein, we investigated the effect and mechanism of m5C RNA modification in the microglia induced CI/RI neuroinflammation. We found that the m5C RNA modification levels decreased in the primary microglia isolated from a mouse model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and the BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), and this change was accompanied by an increase in the M1/M2 polarization ratio. Furthermore, the expression of m5C demethylase TET1 in microglia increased, which promoted M1 polarization but impeded M2 polarization. Mechanistically, the higher TET1 expression decreased the m5C modification level of RelB and enhanced its mRNA stability, which subsequently increased the M1/M2 polarization ratio. In conclusion, this study provides insight into the role of m5C RNA modification in the pathogenesis of cerebral I/R neuroinflammation and may deepen our understanding on clinical therapy targeting the TET1-RelB axis.

Neuron-targeted liposomal coenzyme Q10 attenuates neuronal ferroptosis after subarachnoid hemorrhage by activating the ferroptosis suppressor protein 1/coenzyme Q10 system.

Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. SAH disrupts the blood‒brain barrier, leading to the release of iron ions from blood within the subarachnoid space, subsequently inducing neuronal ferroptosis. A recently discovered protein, known as ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10 by introducing the neuron-targeting peptide Tet1 onto the surface of liposomal CoQ10. Our objective was to determine whether this formulation could activate the FSP1 system and subsequently inhibit neuronal ferroptosis. Our findings revealed that neuron-targeted liposomal CoQ10 effectively localized to neurons at the lesion site after SAH. Furthermore, it facilitated the upregulation of FSP1, reduced the accumulation of malondialdehyde and reactive oxygen species, inhibited neuronal ferroptosis, and exerted neuroprotective effects both in vitro and in vivo. Our study provides evidence that supplementation with CoQ10 can effectively activate the FSP1 system. Additionally, we developed a neuron-targeted liposomal CoQ10 formulation that can be selectively delivered to neurons at the site of SAH. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH. STATEMENT OF SIGNIFICANCE: Subarachnoid hemorrhage (SAH) is primarily attributed to the rupture of intracranial aneurysms and is associated with a high incidence of disability and mortality. Ferroptosis suppressor protein 1 (FSP1), exerts anti-ferroptotic effects by facilitating the conversion of oxidative coenzyme Q 10 (CoQ10) to its reduced form, which effectively scavenges reactive oxygen radicals and mitigates iron-induced ferroptosis. In our investigation, we observed an increase in FSP1 levels following SAH. However, the depletion of CoQ10 caused by SAH hindered the biological function of FSP1. Therefore, we created neuron-targeted liposomal CoQ10. We find that it effectively localized to neurons at the lesion site after SAH and activated the FSP1/CoQ10 system. This innovative approach represents a promising therapeutic strategy for neuronal ferroptosis following SAH and other central nervous system diseases characterized by disruption of the blood-brain barrier.

CRISPR/dCas9-Tet1-Mediated DNA Methylation Editing.

DNA methylation is a key epigenetic mechanism underlying many biological processes, and its aberrant regulation has been tightly associated with various human diseases. Precise manipulation of DNA methylation holds the promise to advance our understanding of this critical mechanism and to develop novel therapeutic methods. Previously, we were only able to alter genome-wide DNA methylation by treating with small molecules (e.g., 5-Aza-2-deoxycytidine) or perturbing relevant genes (e.g., DNA methyltransferase) targetlessly, which makes it challenging to investigate the functional significance of this epigenetic mark at specific genomic loci. By fusing the catalytic domain of a key enzyme in the DNA demethylation process (Ten-eleven translocation dioxygenases 1, Tet1) with a reprogrammable sequence-specific DNA-targeting molecular protein, dCas9, we developed a DNA methylation editing tool (dCas9-Tet1) to demethylate specific genomic loci in a targeted manner. This dCas9-Tet1 system allows us to study the role of DNA methylation at almost any given loci with only the replacement of a single-guide RNA. Here, we describe a protocol that enables modular and scalable manipulation of DNA methylation at specific genomic loci in various cell cultures with high efficiency and specificity using the dCas9-Tet1 system. Key features • Precisely editing the DNA methylation of specific genomic loci in a targeted manner. • Fine-tuning gene expression without changing DNA sequence. • Applicable to many types of cell cultures and with the potential for ex vitro and in vivo applications.

Vitamin C enhances co-localization of novel TET1 nuclear bodies with both Cajal and PML bodies in colorectal cancer cells.

Deregulation of ten-eleven Translocation protein 1 (TET1) is commonly reported to induce imbalances in gene expression and subsequently to colorectal cancer development (CRC). On the other hand, vitamin C (VitC) improves the prognosis of colorectal cancer by reprogramming the cancer epigenome and limiting chemotherapeutic drug resistance events. In this study, we aimed to characterize TET1-specific subcellular compartments and evaluate the effect of VitC on TET1 compartmentalization in colonic tumour cells. We demonstrated that TET1 is concentrated in coarse nuclear bodies (NB) and 5-hydroxymethylcytosine (5hmC) in foci in colorectal cancer cells (HCT116, Caco-2, and HT-29). To our knowledge, this is the first report of a novel intracellular localization profile of TET1 and its demethylation marker, 5hmC, in CRC cells. Interestingly, we found that TET1-NBs frequently interacted with Cajal bodies, but not with promyelocytic leukaemia (PML) bodies. In addition, we report that VitC treatment of HCT116 cells induces 5hmC foci biogenesis and triggers 5hmC marks to form active complexes with nuclear body components, including both Cajal and PML proteins. Our data highlight novel NB-concentrating TET1 in CRC cells and demonstrate that VitC modulates TET1-NBs' interactions with other nuclear structures. These findings reveal novel TET1-dependent cellular functions and potentially provide new insights for CRC management.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells.

BACKGROUND: Genome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs. RESULTS: We show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine. CONCLUSIONS: The results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Expression of Immunotherapy Target PRAME in Cancer Correlates with Histone H3 Acetylation and Is Unrelated to Expression of Methylating (DMNT3A/3B) and Demethylating (TET1) Enzymes.

Background/Objectives: Preferentially expressed antigen in melanoma (PRAME), a member of the cancer testis antigen family, is a promising target for cancer immunotherapy. Understanding the epigenetic mechanisms involved in the regulation of PRAME expression might be crucial for optimizing anti-PRAME treatments. Methods: Three malignancies of different lineages (sinonasal melanoma, testicular seminoma, and synovial sarcoma), in which immunohistochemical (IHC) reactivity for PRAME is a common yet variable feature, were studied. The expression of PRAME, ten-eleven translocation demethylase 1 (TET1), and DNA methyltransferase (DNMT) 3A and 3B were evaluated using immunohistochemistry. Moreover, the expression of two epigenetic marks, 5-hydroxymethylcytosine (5hmC) and histone 3 acetylation (H3ac), was tested. Results: All PRAME-positive tumors expressed medium-to-high levels of H3ac but differed considerably with respect to other markers. In seminomas, PRAME expression correlated with TET1, but in melanomas and synovial sarcomas, it correlated with both DNMTs and DNMT3A, respectively. Conclusions: PRAME expression was not determined by a balance between the global expression of DNA methylating/demethylating enzymes. However, histone acetylation may be one of the epigenetic mechanisms involved in PRAME regulation. Thus, the therapeutic combination of histone deacetylase inhibitors and PRAME immunotherapy merits further investigation.

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

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.

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5.

BACKGROUND: Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC. METHODS: Bioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR. RESULTS: UCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner. CONCLUSION: UCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC.

JMJD3 regulate H3K27me3 modification via interacting directly with TET1 to affect spermatogonia self-renewal and proliferation.

BACKGROUND: In epigenetic modification, histone modification and DNA methylation coordinate the regulation of spermatogonium. Not only can methylcytosine dioxygenase 1 (TET1) function as a DNA demethylase, converting 5-methylcytosine to 5-hydroxymethylcytosine, it can also form complexes with other proteins to regulate gene expression. H3K27me3, one of the common histone modifications, is involved in the regulation of stem cell maintenance and tumorigenesis by inhibiting gene transcription. METHODS: we examined JMJD3 at both mRNA and protein levels and performed Chip-seq sequencing of H3K27me3 in TET1 overexpressing cells to search for target genes and signaling pathways of its action. RESULTS: This study has found that JMJD3 plays a leading role in spermatogonia self-renewal and proliferation: at one extreme, the expression of the self-renewal gene GFRA1 and the proliferation-promoting gene PCNA was upregulated following the overexpression of JMJD3 in spermatogonia; at the other end of the spectrum, the expression of differentiation-promoting gene DAZL was down-regulated. Furthermore, the fact that TET1 and JMJD3 can form a protein complex to interact with H3K27me3 has also been fully proven. Then, through analyzing the sequencing results of CHIP-Seq, we found that TET1 targeted Pramel3 when it interacted with H3K27me3. Besides, TET1 overexpression not only reduced H3K27me3 deposition at Pramel3, but promoted its transcriptional activation as well, and the up-regulation of Pramel3 expression was verified in JMJD3-overexpressing spermatogonia. CONCLUSION: In summary, our study identified a novel link between TET1 and H3K27me3 and established a Tet1-JMJD3-H3K27me3-Pramel3 axis to regulate spermatogonia self-renewal and proliferation. Judging from the evidence offered above, we can safely conclude that this study provides new ideas for further research regarding the mechanism of spermatogenesis and spermatogenesis disorders on an apparent spectrum.

Tet1/DLL3/Notch1 signal pathway affects hippocampal neurogenesis and regulates depression-like behaviour in mice.

Ten-eleven translocation protein 1 (Tet1) is associated with the regulation of depression-like behaviour in mice. However, the mechanism by which Tet1 affects neurogenesis in mice to regulate depression-like behaviours remains unclear. In this study, the chronic social defeat stress (CSDS) paradigm was constructed by overexpressing Tet1 protein in the mouse hippocampus, and 5-ethynyl-2'-deoxyuridine (EdU, 50 mg/kg) was injected on the seventh day to explore the mechanism of the regulation of the Tet1/Delta-like protein 3 (DLL3)/Notch1 protein pathway in mice hippocampal neurogenesis and its influence on depression-like behaviour. Following CSDS, the expression level of Tet1 decreased significantly. Moreover, due to the downregulation of Tet1 protein, the maintenance of the DNA methylation and demethylation balance was affected, resulting in a significant increase in the methylation levels of Notch1 and DLL3 and a significant decrease in the protein expression levels of DLL3, Notch1, and brain-derived neurotrophic factor (BDNF). At the same time, the proliferation and differentiation of neurones were affected, which was related to a significant decrease in the number of EdU+, doublecortin (DCX)+, and Ki67+ cells in the hippocampus of the CSDS model mice. When the Tet1 protein was overexpressed in the mouse hippocampus, DLL3 and Notch1 protein expression levels were upregulated, promoting hippocampal neurogenesis and alleviating depression-like behaviour in mice. These findings suggest that regulation of the hippocampal Tet1/DLL3/Notch1 protein pathway to influence neurogenesis may be a therapeutic strategy for depression.

TET1 inhibits liver fibrosis by blocking hepatic stellate cell activation.

Hepatic stellate cells (HSCs) are critical regulator contributing to the onset and progression of liver fibrosis. Chronic liver injury triggers HSCs to undergo vast changes and trans-differentiation into a myofibroblast HSCs, the mechanism remains to be elucidated. This study investigated that the involvement of hydroxymethylase TET1 (ten-eleven translocation 1) in HSC activation and liver fibrosis. It is revealed that TET1 levels were downregulated in the livers in mouse models of liver fibrosis and patients with cirrhosis, as well as activated HSCs in comparison to quiescent HSCs. In vitro data showed that the inhibition of TET1 promoted the activation HSC, whereas TET1 overexpression inhibited HSC activation. Moreover, TET1 could regulate KLF2 (Kruppel-like transcription factors) transcription by promoting hydroxymethylation of its promoter, which in turn suppressed the activation of HSCs. In vivo, it is confirmed that liver fibrosis was aggravated in Tet1 knockout mice after CCl4 injection, accompanied by excessive activation of primary stellate cells, in contrast to wild-type mice. In conclusion, we suggested that TET1 plays a significant role in HSC activation and liver fibrosis, which provides a promising target for anti-fibrotic therapies.

Locus-Specific and Stable DNA Demethylation at the H19/IGF2 ICR1 by Epigenome Editing Using a dCas9-SunTag System and the Catalytic Domain of TET1.

DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders.