PROSER1 mediates TET2 O-GlcNAcylation to regulate DNA demethylation on UTX-dependent enhancers and CpG islands

DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.

Advances in the DNA methylation hydroxylase TET1

Background The ten-eleven translocation 1 (TET1) protein is a 5-methylcytosine hydroxylase that belongs to the TET protein family of human α-ketoglutarate oxygenases. TET1 recognizes and binds to regions of high genomic 5′-CpG-3′ dinucleotide density, such as CpG islands, initiates the DNA demethylation program, and maintains DNA methylation and demethylation balance to maintain genomic methylation homeostasis and achieve epigenetic regulation. This article reviews the recent research progress of TET1 in the mechanism of demethylation, stem cells and immunity, various malignant tumours and other clinical diseases. Conclusion TET1 acts as a key factor mediating demethylation, the mechanism of which still remains to be investigated in detail. TET1 is also critical in maintaining the differentiation pluripotency of embryonic stem cells and plays anti- or oncogenic roles in combination with different signalling pathways in different tumours. In certain tumours, its role is still controversial. In addition, the noncatalytic activity of TET1 has gradually attracted attention and has become a new direction of research in recent years.

Abstract MP245: Tet Proteins Regulate Second Heart Field Multipotent Progenitors Differentiating To Myocytes

DNA methylation and demethylation play an important role in shaping the epigenetic landscape and chromatin accessibility to control gene expression during development in mammals. Ten-eleven Translocation (Tet1, Tet2 and Tet3) is a family of dioxygenases that catalyze DNA methylation oxidation with ultimate DNA demethylation. Our previous study showed that cardiac-specific deletion of Tet2 and Tet3 could disrupt YY1-mediated long range chromatin interactions during heart development and lead to ventricular non-compaction cardiomyopathy. However, it is still unclear whether and how Tet protein mediated epigenetic modifications contribute to cardiac lineage specification during embryonic development. In this study, we generated cardiac specific Tet1-3 triple deficient (Tet-TKO) mouse lines using various cardiac specific Cres to evaluate the function of Tet protein in regulating cardiac lineage specification. We observed developmental defects at outflow tract (OFT) in Tet-TKO embryos, suggesting that Tet deficiency affects the second heart field (SHF) development. Single cell RNA-seq analysis further revealed the accumulation of multipotent SHF progenitors and subsequent halt of myocyte differentiation upon Tet depletion. At the molecular level, we found that Tet ablation perturbs the transcriptional network of Islet1, a transcription factor that is crucial for cardiac development in embryos. Overall, our study demonstrates a critical role of Tet-mediated epigenetic regulation for embryonic cardiac development.

Role of Tet2 in Regulating Adaptive and Innate Immunity

Accumulated evidence indicates that epigenetic modifications play central roles in gene expression regulation and participate in developing many autoimmune and autoinflammatory diseases. Mechanistically, epigenetic modifications act as a bridge between environmental and cellular factors and susceptibility genes. DNA methylation is a critical epigenetic modification that is regulated by ten-eleven translocation (TET) enzymes. Accumulating evidence has revealed that TET family proteins function as gene regulators and antitumor drug targets mainly because of their ability to oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Recently, the effect of Tet2, an essential TET protein, on the development of autoimmune diseases has been explored. In this review, we summarize the current understanding of Tet2 in immune response regulation, clarify the mechanisms of Tet2 in B and T cell differentiation and function, and discuss the opposing effects of Tet2 on inflammatory gene expression in the immune system to provide new potential therapeutic targets for related diseases.

TET is targeted for proteasomal degradation by the PHD-pVHL pathway to reduce DNA hydroxymethylation

Hypoxia-inducible factors are heterodimeric transcription factors that play a crucial role in a cell's ability to adapt to low oxygen. The von Hippel-Lindau tumor suppressor (pVHL) acts as a master regulator of HIF activity, and its targeting of prolyl hydroxylated HIF-α for proteasomal degradation under normoxia is thought to be a major mechanism for pVHL tumor suppression and cellular response to oxygen. Whether pVHL regulates other targets through a similar mechanism is largely unknown. Here, we identify TET2/3 as novel targets of pVHL. pVHL induces proteasomal degradation of TET2/3, resulting in reduced global 5-hydroxymethylcytosine levels. Conserved proline residues within the LAP/LAP-like motifs of these two proteins are hydroxylated by the prolyl hydroxylase enzymes (PHD2/EGLN1 and PHD3/EGLN3), which is prerequisite for pVHL-mediated degradation. Using zebrafish as a model, we determined that global 5-hydroxymethylcytosine levels are enhanced in vhl-null, egln1a/b-double-null, and egln3-null embryos. Therefore, we reveal a novel function for the PHD-pVHL pathway in regulating TET protein stability and activity. These data extend our understanding of how TET proteins are regulated and provide new insight into the mechanisms of pVHL in tumor suppression.

Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naïve pluripotency marker

Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

Dysregulation of the TET family of epigenetic regulators in lymphoid and myeloid malignancies

TET2 is frequently mutated in lymphoid and myeloid malignancy. Lio et al review the current understanding of the role of TET enzymes in lymphoid and myeloid malignancy, highlighting that loss of TET protein function can occur either by mutation or as a result of metabolic alteration.

Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to differences in gene expression

Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.