TET1 Catalytic Domain Research Articles

Abstract 7549: Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1

Abstract DNA methylation inhibitors such as 5-azacytidine (Aza) and 5-aza-2′-deoxycytidine have been extensively used in patients with hematologic disorders and other neoplasms since their approval by the FDA. However, global DNA demethylating agents can result in intolerable toxicities; therefore, targeted epigenetic modulation is needed. Here, we searched for epigenetic changes with adaptive resistance to anti-VEGF antibody (AVA) in the TME and a novel strategy for targeted demethylation. Methods: We examined methylation and transcriptional changes in orthotopic ovarian cancer models with emergence of AVA resistance. We developed a targeted demethylation strategy for BARD1 to enhance the effectiveness of AVA. We used a novel dCas9-CRISPR-based system for targeted BARD1 CpG demethylation (derived by fusing the deactivated Cas9 [dCas9] with the TET1 catalytic domain) delivered in vivo using a nanoliposomal carrier. Results: We identified BARD1 methylation and reduced expression as lead candidate with AVA resistance; multiple CpG island sites in the BARD1 gene from adaptive AVA-resistant tumors were methylated. Next, we investigated the biological effects of global and targeted DNA methylation. Azacytidine along with AVA therapy was tested in a series of models (fibrosarcoma, PDX and orthotopic ovarian cancer models) and resulted in reduced tumor burden compared with the control and monotherapy groups (p<0.01). Next, to determine whether targeted demethylated BARD1 would have antitumor effects in vivo, we used an orthotopic SKOV3ip1luc ovarian cancer mouse model. Mice were treated with DOPC-dCas9-TET1-sgcontrol- (control) and DOPC-dCas9-TET1-sgBARD1 (sgBARD1). Tumor weight in the sgBARD1-3 group was significantly (p<0.01) reduced compared with the control group with no significant change in mouse body weight. We found that BARD1 expression was significantly increased in sgBARD1-treated group compared with control (p<0.001). Next, to determine whether demethylated BARD1 would combine well with AVA therapy in the SKOV3ip1 model, we combined sgBARD1-3 with bevacizumab (bev); sgBARD1-3 alone led to a significant decrease in tumor burden compared with control groups. Moreover, tumor burden was significantly reduced in the sgBARD1-3 and bevacizumab combination group compared with the sgcontrol group and bev alone group. BARD1 expression was significantly increased in sgBARD1-3-treated and in combination tumor samples compared with controls. To check for potential off-target effects, we tested the efficacy of another sgBARD1 and found that tumor burden in the sgBARD1-1 group was significantly reduced compared with control group. Conclusion: Our novel targeted approach results in robust antitumor effects when combined with AVA therapy in multiple cancer models. Collectively, our findings provide a novel approach for gene targeted demethylation and could have implications for epigenetic therapies. Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Elaine Stur, Sudhir Kumar, Lingegowda S. Mangala, Nazende Nur Bayram, Pahul Hanjra, Sara Corvigno, Stephen Baylin, Gabriel Lopez-Berestein, Anil K. Sood. Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7549.

510-P: Tissue Nanotransfection–Based Endothelial-Targeted Epigenetic Gene Editing In Vivo to Rescue Diabetic Ischemic Wounds

Our previous work (PMID: 35192691) has identified that genetically silenced phospholipase Cγ2 (PLCγ2) hinders VEGF therapy of the diabetic ischemic limb. Hyperglycemia caused hypermethylation of endothelial specific gene promoter. Given that epigenetic changes are reversible, this work tests the significance of gene-targeted therapeutic DNA demethylation in improving blood flow to diabetic ischemic wounds. Bipedicle ischemic wounds were placed in streptozotocin (STZ) induced acute diabetic C57BL6 mice. Diabetic VWF+ dermal endothelial cells were isolated using flow sorting. In wound tissue, PLCγ2 promoter CpG methylation levels were analyzed using bisulfite sequencing. Next, to specifically demethylate PLCγ2 promoter in endothelial cells, a demethylation cocktail was designed: (i) (scFv)-TET1 catalytic domain (TET1CD) system capable of inducing targeted DNA methylation, and (ii) endothelial promoter driven gene specific guide RNAs. This demethylation cocktail was delivered at the diabetic ischemic wound-edge employing non-viral topical tissue nano-transfection (TNT) technology. PLCγ2 protein expression outcomes were assessed using flow cytometry. Functional outcome of such demethylation was assessed using Laser Speckle Perfusion imaging (Perimed Inc.) and ultrasonography (Vevo 2100) at days 3, 7&10. Overall DNA hyper methylation was prominent in murine ischemic flaps as demonstrated by the increased ratio of 5-methylcytosine (methylation mark) to 5-hydroxymethylcytosine (demethylation mark) (n=5). Specifically, the PLCγ2 promoter was hypermethylated (n=5). TNT mediated endothelial-targeted demethylation of the PLCγ2 promoter increased its expression (n=4). Such demethylation-based upregulation of PLCγ2 improved wound tissue blood flow with increased abundance of VWF+/PLCG2+ vascular elements (n=4). TNT-based endothelial demethylation of the PLCγ2 gene promoter improved perfusion of cutaneous diabetic wounds. Disclosure S.S.Verma: None. S.Kacar: None. S.C.Gnyawali: None. L.S.Fehme: None. C.K.Sen: n/a. K.Singh: None. Funding U.S. Department of Defense (W81XWH-22-1-0146)

Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis.

CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.

Ten-Eleven Translocation Ablation Impairs Cardiac Differentiation of Mouse Embryonic Stem Cells.

Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.

Suppression of DNA Double-Strand Break Formation by DNA Polymerase β in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons.

Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase β (Polβ) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polβ fl/fl mice of either sex, in which forebrain postmitotic excitatory neurons lack Polβ expression. Polβ deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polβ maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENT Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polβ, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polβ deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

TET1 promotes growth of T-cell acute lymphoblastic leukemia and can be antagonized via PARP inhibition.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by skewed epigenetic patterns, raising the possibility of therapeutically targeting epigenetic factors in this disease. Here we report that among different cancer types, epigenetic factor TET1 is highly expressed in T-ALL and is crucial for human T-ALL cell growth in vivo. Knockout of TET1 in mice and knockdown in human T cell did not perturb normal T-cell proliferation, indicating that TET1 expression is dispensable for normal T-cell growth. The promotion of leukemic growth by TET1 was dependent on its catalytic property to maintain global 5-hydroxymethylcytosine (5hmC) marks, thereby regulate cell cycle, DNA repair genes, and T-ALL associated oncogenes. Furthermore, overexpression of the Tet1-catalytic domain was sufficient to augment global 5hmC levels and leukemic growth of T-ALL cells in vivo. We demonstrate that PARP enzymes, which are highly expressed in T-ALL patients, participate in establishing H3K4me3 marks at the TET1 promoter and that PARP1 interacts with the TET1 protein. Importantly, the growth related role of TET1 in T-ALL could be antagonized by the clinically approved PARP inhibitor Olaparib, which abrogated TET1 expression, induced loss of 5hmC marks, and antagonized leukemic growth of T-ALL cells, opening a therapeutic avenue for this disease.

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.

Hypercholesterolemia Accelerates the Aging Phenotypes of Hematopoietic Stem Cells by a Tet1-Dependent Pathway

Hypercholesterolemia accelerates the phenotypes of aging in hematopoietic stem cells (HSCs). As yet, little is known about the underlying mechanism. We found that hypercholesterolemia downregulates Ten eleven translocation 1 (Tet1) in HSCs. The total HSC population was increased, while the long-term (LT) population, side population and reconstitution capacity of HSCs were significantly decreased in Tet1−/− mice. Expression of the Tet1 catalytic domain in HSCs effectively restored the LT population and reconstitution capacity of HSCs isolated from Tet1−/− mice. While Tet1 deficiency upregulated the expression of p19 and p21 in HSCs by decreasing the H3K27me3 modification, the restoration of Tet1 activity reduced the expression of p19, p21 and p27 by restoring the H3K27me3 and H3K36me3 modifications on these genes. These results indicate that Tet1 plays a critical role in maintaining the quiescence and reconstitution capacity of HSCs and that hypercholesterolemia accelerates HSC aging phenotypes by decreasing Tet1 expression in HSCs.

Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways

Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.

Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity.

The ten-eleven translocation (TET) protein family, consisting of three isoforms (TET1/2/3), have been found in mammalian cells and have a crucial role in 5-methylcytosine demethylation in genomic DNA through the catalysis of oxidation reactions assisted by 2-oxoglutarate (2OG). DNA methylation/demethylation contributes to the regulation of gene expression at the transcriptional level, and recent studies have revealed that TET1 is highly elevated in malignant cells of various diseases and related to malignant alteration. TET1 inhibitors based on a scaffold of thioether macrocyclic peptides, which have been discovered by the random nonstandard peptide integrated discovery (RaPID) system, are reported. The affinity-based selection was performed against the TET1 compact catalytic domain (TET1CCD) to yield thioether macrocyclic peptides. These peptides exhibited inhibitory activity of the TET1 catalytic domain (TET1CD), with an IC50 value as low as 1.1 μm. One of the peptides, TiP1, was also able to inhibit TET1CD over TET2CD with tenfold selectivity, although it was likely to target the 2OG binding site; this provides a good starting point to develop more selective inhibitors.

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF-TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF-TET1cd fusions, the SunTag-TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons.

TET1 deficiency attenuates the DNA damage response and promotes resistance to DNA damaging agents

ABSTRACTRecent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G2/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.

Immunofluorescence Imaging Strategy for Evaluation of the Accessibility of DNA 5-Hydroxymethylcytosine in Chromatins.

DNA 5-hydroxymethylcytosine (5hmC) is an important epigenetic modification found in various mammalian cells. Immunofluorescence imaging analysis essentially provides visual pictures for the abundance and distribution of DNA 5hmC in single cells. However, nuclear DNA is usually wrapped around nucleosomes, packaged into chromatins, and further bound with many functional proteins. These physiologically relevant events would generate barriers to the anti-5hmC antibody to selectively recognize 5hmC in DNA. By taking advantage of these naturally generated barriers, here, we present a strategy to evaluate the accessibility of DNA 5hmC in chromatins in situ. We demonstrate that a few of the 5hmC sites in DNA are exposed or accessible to anti-5hmC antibody under nondenaturing conditions, suggesting that these 5hmC sites are not covered by functional DNA-binding proteins in mouse embryonic stem cells. Consistently, these 5hmC foci were distributed in open euchromatin regions as revealed by the 4',6-diamidino-2-phenylindole (DAPI) staining. By overexpressing TET1 catalytic domain (responsible for oxidation 5mC to produce 5hmC) in human MCF-7 cells, we observed a significant increase in accessible 5hmC along with an increase in total 5hmC sites. Collectively, by the use of the nondenaturing immunofluorescence imaging approach, we could obtain a visual landscape on the accessibility of DNA 5hmC in chromatins.

Ten-eleven translocation 1 functions as a mediator of SOD3 expression in human lung cancer A549 cells

Superoxide dismutase (SOD) 3, one of the SOD isozymes, plays a pivotal role in extracellular redox homeostasis. The expression of SOD3 is regulated by epigenetics in human lung cancer A549 cells and human monocytic THP-1 cells; however, the molecular mechanisms governing SOD3 expression have not been elucidated in detail. Ten-eleven translocation (TET), a dioxygenase of 5-methylcytosine (5mC), plays a central role in DNA demethylation processes and induces target gene expression. In the present study, TET1 expression was abundant in U937 cells, but its expression was weakly expressed in A549 and THP-1 cells. These results are consistent with the expression pattern of SOD3 and its DNA methylation status in these cells. Moreover, above relationship was also observed in human breast cancer cells, human prostate cancer cells, and human skin fibroblasts. The overexpression of TET1-catalytic domain (TET1-CD) induced the expression of SOD3 in A549 cells, and this was accompanied by the direct binding of TET1-CD to the SOD3 promoter region. Furthermore, in TET1-CD-transfected A549 cells, the level of 5-hydroxymethylcytosine within that region was significantly increased, whereas the level of 5mC was decreased. The results of the present study demonstrate that TET1 might function as one of the key molecules in SOD3 expression through its 5mC hydroxylation in A549 cells.

Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

Abstract 4432: Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation

Abstract DNA demethylating agents are useful therapeutic drugs for human cancer; 5-aza-2’-deoxycytidine and 5-azacytidine for myelodysplastic syndrome are good examples. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects. However, precise mechanisms for anticancer activity of these demethylating reagents have yet to be established, mainly due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulates more genes than ones expressing TET1-CDwt without MBD (TET1-CDwt) or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site (TSS). Interestingly, 65% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with 5-azacytidine, DNA demethylating agent. These results suggested a fact that gene reactivation by both methods was primarily based on DNA demethylation. In order to examine growth inhibitory effects of DNA demethylation to cancer cells, we utilized a tetracycline inducible system to regulate the expression of MBD-TET1-CDwt using a prostate cancer cell line. Induction of MBD-TET1-CDwt demethylated and upregulated the glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells. Flow cytometry analysis showed decreased cells in S phase and increased cells in G1 phase by MBD-TET1-CDwt expression. Our present results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation, and that our methods allow us to analyze the effects of global DNA demethylation in a wide variety of cancer cells. Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Kanchan Chakma, Yuriko Saiki, Akira Horii. Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4432.

Epigenetic inactivation of the CpG demethylase TET1 as a DNA methylation feedback loop in human cancers.

Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis.

A CRISPR-based approach for targeted DNA demethylation.

In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.

5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme.

5-hydroxymethylcytosine is a new epigenetic modification deriving from the oxidation of 5-methylcytosine by the TET hydroxylase enzymes. DNA hydroxymethylation drives DNA demethylation events and is involved in the control of gene expression. Deregulation of TET enzymes causes developmental defects and is associated with pathological conditions such as cancer. Little information thus far is available on the regulation of TET activity by post-translational modifications. Here we show that TET1 protein is able to interact with PARP-1/ARTD1 enzyme and is target of both noncovalent and covalent PARylation. In particular, we have demonstrated that the noncovalent binding of ADP-ribose polymers with TET1 catalytic domain decreases TET1 hydroxylase activity while the covalent PARylation stimulates TET1 enzyme. In addition, TET1 activates PARP-1/ARTD1 independently of DNA breaks. Collectively, our results highlight a complex interplay between PARylation and TET1 which may be helpful in coordinating the multiple biological roles played by 5-hydroxymethylcytosine and TET proteins.