CXXC Domain Research Articles

Lysine Demethylase KDM2A Promotes Proteasomal Degradation of TCF/LEF Transcription Factors in a Neddylation-Dependent Manner.

Canonical Wnt signaling is essential for a plethora of biological processes ranging from early embryogenesis to aging. Malfunctions of this crucial signaling pathway are associated with various developmental defects and diseases, including cancer. Although TCF/LEF transcription factors (TCF/LEFs) are known to be essential for this pathway, the regulation of their intracellular levels is not completely understood. Here, we show that the lysine demethylase KDM2A promotes the proteasomal destabilization of TCF/LEFs independently of its demethylase domain. We found that the KDM2A-mediated destabilization of TCF/LEFs is dependent on the KDM2A zinc finger CXXC domain. Furthermore, we identified the C-terminal region of TCF7L2 and the CXXC domain of KDM2A as the domains responsible for the interaction between the two proteins. Our study is also the first to show that endogenous TCF/LEF proteins undergo KDM2A-mediated proteasomal degradation in a neddylation-dependent manner. Here, we reveal a completely new mechanism that affects canonical Wnt signaling by regulating the levels of TCF/LEF transcription factors through their KDM2A-promoted proteasomal degradation.

Aberrant Splicing of MBD1 Reshapes the Epigenome to Drive Convergent Myeloerythroid Defects in MDS

Splicing defects are a characteristic feature of myelodysplastic syndromes (MDS) and typically associate with specific recurrent splicing factor mutations. However, a subset of transcripts exhibit abnormal splicing regardless of mutational background, occurring even in the absence of splicing-related mutations. These shared splicing events likely include common underlying drivers of MDS hematopoietic defects, yet the functions of the resulting transcripts remain unknown. We identified a long isoform of Methyl-CpG-Binding Domain 1 ( MBD1) as the product of one such mutation-independent splicing event. To understand whether altered MBD1 splicing contributes to hematopoietic dysfunction, we overexpressed isoforms of MBD1 in cord blood CD34+ cells and found that the MDS-associated full-length isoform (MBD1-L), containing MBD1's 3rd CXXC domain, impaired erythroid differentiation by stalling cell cycling and promoting apoptosis. In contrast, the MBD1-ΔCXXC3 isoform (MBD1-S), preferentially produced in healthy cells, did not induce these defects. Recapitulating these findings, the MBD1-L isoform uniquely impaired reconstitution capacity in vivo, particularly in the erythroid and myeloid lineages, and in addition produced an enrichment of the MDS transcriptomic signature on RNA-seq profiling. The unique CXXC3 domain of MBD1-L specifically binds non-methylated CpGs, and exhibits greater target affinity than the shared MBD domain, which is responsible for mCpG binding. Given MBD1's key role in heterochromatin maintenance at mCpG regions, we hypothesized that the unique function of MBD1-L in MDS may be attributable to the refocusing of MBD1-mediated epigenetic repression from canonical, methylated DNA sites to unmethylated sites. Isoform-specific CUT&RUN and multi-omics profiling in cord blood CD34+ cells revealed that the inclusion of the CXXC3 exon triggers a striking redistribution of MBD1 from gene bodies and intergenic regions to hypomethylated promoter CpG islands, resulting in widespread suppression of promoter chromatin accessibility and downregulation of cell-cycle-related transcripts. Characterization of the MBD1 interactome by rapid immunoprecipitation mass spectrometry showed that the MBD1-L isoform preferentially associates with the SETDB1:ATF7IP H3K9 methylator complex, supporting active heterochromatin establishment at MBD1-bound promoters. Among the direct targets uniquely repressed by MBD1-L is BCOR, a recurrent LOF gene in MDS whose loss perturbs hematopoietic differentiation and promotes self-renewal. Downregulation of BCOR by MBD1-L led to the derepression of BCOR-controlled genes, contributing to an enriched stem cell-associated transcriptomic signature resembling BCOR LOF. To investigate whether reversal of MBD1-L splicing can restore hematopoietic output in diseased cells, we delivered lipid-encapsulated splice-switching antisense oligonucleotides targeting the CXXC3 exon into primary human MDS cells, and observed an increase in differentiation in vitro. In addition, depletion of MBD1-L in the MDSL cell line using isoform-specific shRNAs increased cell proliferation, confirming that targeted reduction of MBD1-L inverted the quiescent, differentiation-impaired phenotype imposed by its overexpression. Our results demonstrate that MBD1 isoforms act on separate compartments of genomic sequence to carry out divergent functions in hematopoietic cells, and that disease-associated overproduction of MBD1-L compromises hematopoietic output and lineage differentiation. These findings provide the first evidence that mutation-independent splicing changes can drive hematopoietic dysfunction in MDS.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Insertion of the CXXC domain of KMT2A into YAP1: An unusual mechanism behind the formation of a chimeric oncogenic protein.

Most neoplasia-associated gene fusions are formed through the fusion of the 5'-part of one gene with the 3'-part of another. We here describe a unique mechanism, by which a part of the KMT2A gene through an insertion replaces part of the YAP1 gene. The resulting YAP1::KMT2A::YAP1 (YKY) fusion was verified by RT-PCR in three cases of sarcoma morphologically resembling sclerosing epithelioid fibrosarcoma (SEF-like sarcoma). In all cases, a portion (exons 4/5-6) encoding the CXXC domain of KMT2A was inserted between exon 4/5 and exon 8/9 of YAP1. The inserted sequence from KMT2A thus replaced exons 5/6-8 of YAP1, which encode an important regulatory sequence of YAP1. To evaluate the cellular impact of the YKY fusion, global gene expression profiles from fresh frozen and formalin-fixed YKY-expressing sarcomas were compared with control tumors. The effects of the YKY fusion, as well as YAP1::KMT2A and KMT2A::YAP1 fusion constructs, were further studied in immortalized fibroblasts. Analysis of differentially upregulated genes revealed significant overlap between tumors and cell lines expressing YKY, as well as with previously reported YAP1 fusions. Pathway analysis of upregulated genes in cells and tumors expressing YKY revealed an enrichment of genes included in key oncogenic signaling pathways, such as Wnt and Hedgehog. As these pathways are known to interact with YAP1, it seems likely that the pathogenesis of sarcomas with the YKY fusion is linked to distorted YAP1 signaling.

Back Cover

Chinese Journal of ChemistryVolume 41, Issue 10 p. 1288-1288 Back CoverFree Access Back Cover First published: 15 April 2023 https://doi.org/10.1002/cjoc.202390105AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract The cover picture shows TET3 CXXC domain searching CpG motifs with preferences on their flanking DNA sequences, which is profiled at the single-molecule limit. The selective binding dynamics of a CXXC on a gene facilitates our understanding to epigenetic information reading mechanisms. More details are discussed in the article by Yu et al. on page 1177—1184. Volume41, Issue1015 May, 2023Pages 1288-1288 RelatedInformation

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling†

Comprehensive SummaryRecognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development. The TET3 CXXC domain binds to CpG DNA, serving a basic epigenetic information reading mechanism. During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence, the protein‐DNA interactions are beyond CpG dinucleotide. However, the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploited, which is hard for ensemble methods to probe. Here, we used single‐molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA. Our single‐molecule binding profile revealed that CXXC‐DNA interactions involve both CpG motifs and their flanking sequences. The residence time of TET3 CXXC differs by about 1000 times in five distinguished CpG clusters in the context of a CpG island. Moreover, we performed multi‐state hidden Markov modeling analysis on the zipping/unzipping dynamics of a CpG hairpin, discovering TET3 CXXC's preference on CpG motifs regarding the –2 to +2 flanking bases. Our results shed light on the selective binding dynamics of a CXXC on a gene sequence, facilitating studies on epigenetic information reading mechanisms.

Clustered PHD domains in KMT2/MLL proteins are attracted by H3K4me3 and H3 acetylation-rich active promoters and enhancers

Histone lysine-specific methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukemia (MLL1-4) proteins, mediate positive transcriptional memory. Acting as the catalytic subunits of human COMPASS-like complexes, KMT2A-D methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered (multiple) PHDs localize to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His). The mutations cause a domain loss-of-function. Taken together, our data suggest that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.Graphical

Regulation of Αlpha-Synuclein Gene (SNCA) by Epigenetic Modifier TET1 in Parkinson Disease.

Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression. The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression. In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls. We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

Structural basis for activation of DNMT1

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved α-helix which engages a crucial “Toggle” pocket, displacing a previously described inhibitory linker, and allowing the DNA Recognition Helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature

PurposePathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. MethodsThrough international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. ResultsWe recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. ConclusionPathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.

Small-Molecule Inhibitors of the MLL1 CXXC Domain, an Epigenetic Reader of DNA Methylation.

The CXXC domain is a reader of DNA methylation which preferentially binds to unmethylated CpG DNA motifs. Chromosomal translocations involving the MLL1 gene produce in-frame fusion proteins in which the N-terminal portion of the MLL1 protein harboring its CXXC domain is fused to the C-terminal portion of multiple partners. For the MLL-AF9 fusion, mutations which disrupt CXXC domain-DNA binding abrogate the ability to cause leukemia in mice. Based on this, we initiated an effort to develop small-molecule inhibitors of the MLL1 CXXC domain as a novel approach to therapy. We developed a fluorescence polarization-based assay for MLL CXXC domain-DNA binding and screened a library of Cys-reactive molecules. For the most potent hit from this screen, we have synthesized a library of analogs to explore the structure-activity relationship, defined the binding site using chemical shift perturbations in NMR spectra, and explored the selectivity of compounds across the CXXC domain family.

Ten-Eleven Translocation-3 CXXC domain is critical for postfertilization demethylation and expression of pluripotency genes in pig embryos†.

Enzymes of the ten-eleven translocation family are considered to play an important role in the regulation of DNA methylation patterns by converting 5-methylcytosine to 5-hydroxymethylcytosine. Known as a maternal transcript enriched in mature oocytes, ten-eleven translocation-3 (TET3) has been suggested to initiate DNA demethylation of the paternal genome in zygotes. Previous studies in mouse cells indicate that the N-terminal CXXC domain of TET3 is important in catalyzing the oxidation of 5-methylcytosine through its potential DNA binding ability; however, it is not clear whether the DNA binding capacity of CXXC domain is required for the 5-hydroxymethylcytosine conversion in mammalian embryos. Here, we identified TET3 isoforms in porcine oocytes and investigated the role of the oocyte specific TET3 isoform (pTET3L) in controlling postfertilization demethylation in porcine embryos. The pTET3L possessed sequences representing a known DNA binding domain, the CXXC, and injection of the TET3 CXXC fused with GFP into mature porcine oocytes resulted in exclusive localization of the GFP-CXXC in the pronuclei. The CXXC overexpression reduced the 5-methylcytosine level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, there was an increase in the transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes. Targeted knockdown of pTET3L resulted in the downregulation of pluripotency genes in subsequently developed blastocysts. The findings indicate that the CXXC domain of TET3 serves as a critical component for the postfertilization demethylation of porcine embryos and coordinates proper expression of pluripotency related genes in blastocysts.

Missense variants causing Wiedemann-Steiner syndrome preferentially occur in the KMT2A-CXXC domain and are accurately classified using AlphaFold2.

Wiedemann-Steiner syndrome (WDSTS) is a neurodevelopmental disorder caused by de novo variants in KMT2A, which encodes a multi-domain histone methyltransferase. To gain insight into the currently unknown pathogenesis of WDSTS, we examined the spatial distribution of likely WDSTS-causing variants across the 15 different domains of KMT2A. Compared to variants in healthy controls, WDSTS variants exhibit a 61.9-fold overrepresentation within the CXXC domain-which mediates binding to unmethylated CpGs-suggesting a major role for this domain in mediating the phenotype. In contrast, we find no significant overrepresentation within the catalytic SET domain. Corroborating these results, we find that hippocampal neurons from Kmt2a-deficient mice demonstrate disrupted histone methylation (H3K4me1 and H3K4me3) preferentially at CpG-rich regions, but this has no systematic impact on gene expression. Motivated by these results, we combine accurate prediction of the CXXC domain structure by AlphaFold2 with prior biological knowledge to develop a classification scheme for missense variants in the CXXC domain. Our classifier achieved 92.6% positive and 92.9% negative predictive value on a hold-out test set. This classification performance enabled us to subsequently perform an in silico saturation mutagenesis and classify a total of 445 variants according to their functional effects. Our results yield a novel insight into the mechanistic basis of WDSTS and provide an example of how AlphaFold2 can contribute to the in silico characterization of variant effects with very high accuracy, suggesting a paradigm potentially applicable to many other Mendelian disorders.

The N-terminal domain of TET1 promotes the formation of dense chromatin regions refractory to transcription.

TET (ten-eleven translocation) enzymes initiate active cytosine demethylation via the oxidation of 5-methylcytosine. TET1 is composed of a C-terminal domain, which bears the catalytic activity of the enzyme, and a N-terminal region that is less well characterized except for the CXXC domain responsible for the targeting to CpG islands. While cytosine demethylation induced by TET1 promotes transcription, this protein also interacts with chromatin-regulating factors that rather silence this process, the coordination between these two opposite functions of TET1 being unclear. In the present work, we uncover a new function of the N-terminal part of the TET1 protein in the regulation of the chromatin architecture. This domain of the protein promotes the establishment of a compact chromatin architecture displaying reduced exchange rate of core histones and partial dissociation of the histone linker. This chromatin reorganization process, which does not rely on the CXXC domain, is associated with a global shutdown of transcription and an increase in heterochromatin-associated histone epigenetic marks. Based on these findings, we propose that the dense chromatin organization generated by the N-terminal domain of TET1 could contribute to restraining the transcription enhancement induced by the DNA demethylation activity of this enzyme.

Domain Structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA Methyltransferases.

In mammals, three major DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, have been identified. Dnmt3a and Dnmt3b are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. Dnmt3-like (Dnmt3l), which is a member of the Dnmt3 family but does not possess DNA methylation activity, was reported to be indispensable for global methylation in germ cells. Once the DNA methylation patterns are established, maintenance-type DNA methyltransferase Dnmt1 faithfully propagates them to the next generation via replication. All Dnmts possess multiple domains. For instance, Dnmt3a and Dnmt3b each contain a Pro-Trp-Trp-Pro (PWWP) domain that recognizes the histone H3K36me2/3 mark, an Atrx-Dnmt3-Dnmt3l (ADD) domain that recognizes unmodified histone H3 tail, and a catalytic domain that methylates CpG sites. Dnmt1 contains an N-terminal independently folded domain (NTD) that interacts with a variety of regulatory factors, a replication foci-targeting sequence (RFTS) domain that recognizes the histone H3K9me3 mark and H3 ubiquitylation, a CXXC domain that recognizes unmodified CpG DNA, two tandem Bromo-Adjacent-homology (BAH1 and BAH2) domains that read the H4K20me3 mark with BAH1, and a catalytic domain that preferentially methylates hemimethylated CpG sites. In this chapter, the structures and functions of these domains are described.

A prelude to the proximity interaction mapping of CXXC5

CXXC5 is a member of the zinc-finger CXXC family proteins that interact with unmodified CpG dinucleotides through a conserved ZF-CXXC domain. CXXC5 is involved in the modulation of gene expressions that lead to alterations in diverse cellular events. However, the underlying mechanism of CXXC5-modulated gene expressions remains unclear. Proteins perform their functions in a network of proteins whose identities and amounts change spatiotemporally in response to various stimuli in a lineage-specific manner. Since CXXC5 lacks an intrinsic transcription regulatory function or enzymatic activity but is a DNA binder, CXXC5 by interacting with proteins could act as a scaffold to establish a chromatin state restrictive or permissive for transcription. To initially address this, we utilized the proximity-dependent biotinylation approach. Proximity interaction partners of CXXC5 include DNA and chromatin modifiers, transcription factors/co-regulators, and RNA processors. Of these, CXXC5 through its CXXC domain interacted with EMD, MAZ, and MeCP2. Furthermore, an interplay between CXXC5 and MeCP2 was critical for a subset of CXXC5 target gene expressions. It appears that CXXC5 may act as a nucleation factor in modulating gene expressions. Providing a prelude for CXXC5 actions, our results could also contribute to a better understanding of CXXC5-mediated cellular processes in physiology and pathophysiology.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Dynamics and inhibition of MLL1 CXXC domain on DNA revealed by single-molecule quantification

CpG islands recruit MLL1 via the CXXC domain to modulate chromatin structure and regulate gene expression. The amino acid motif of CXXC also plays a pivotal role in MLL1’s structure and function and serves as a target for drug design. In addition, the CpG pattern in an island governs spatially dependent collaboration among CpGs in recruiting epigenetic enzymes. However, current studies using short DNA fragments cannot probe the dynamics of CXXC on long DNA with crowded CpG motifs. Here, we used single-molecule magnetic tweezers to examine the binding dynamics of MLL1’s CXXC domain on a long DNA with a CpG island. The mechanical strand separation assay allows profiling of protein-DNA complexes and reports force-dependent unfolding times. Further design of a hairpin detector reveals the unfolding time of individual CXXC-CpG complexes. Finally, in a proof of concept we demonstrate the inhibiting effect of dimethyl fumarate on the CXXC-DNA complexes by measuring the dose response curve of the unfolding time. This demonstrates the potential feasibility of using single-molecule strand separation as a label-free detector in drug discovery and chemical biology.

TETology: Epigenetic Mastermind in Action.

Cytosine methylation is a well-explored epigenetic modification mediated by DNA methyltransferases (DNMTs) which are considered "methylation writers"; cytosine methylation is a reversible process. The process of removal of methyl groups from DNA remained unelucidated until the discovery of ten-eleven translocation (TET) proteins which are now considered "methylation editors." TET proteins are a family of Fe(II) and alpha-ketoglutarate-dependent 5-methyl cytosine dioxygenases-they convert 5-methyl cytosine to 5-hydroxymethyl cytosine, and to further oxidized derivatives. In humans, there are three TET paralogs with tissue-specific expression, namely TET1, TET2, and TET3. Among the TETs, TET2 is highly expressed in hematopoietic stem cells where it plays a pleiotropic role. The paralogs also differ in their structure and DNA binding. TET2 lacks the CXXC domain which mediates DNA binding in the other paralogs; thus, TET2 requires interactions with other proteins containing DNA-binding domains for effectively binding to DNA to bring about the catalysis. In addition to its role as methylation editor of DNA, TET2 also serves as methylation editor of RNA. Thus, TET2 is involved in epigenetics as well as epitranscriptomics. TET2 mutations have been found in various malignant hematological disorders like acute myeloid leukemia, and non-malignant hematological disorders like myelodysplastic syndromes. Increasing evidence shows that TET2 plays an important role in the non-hematopoietic system as well. Hepatocellular carcinoma, gastric cancer, prostate cancer, and melanoma are some non-hematological malignancies in which a role of TET2 has been implicated. Loss of TET2 is also associated with atherosclerotic vascular lesions and endometriosis. The current review elaborates on the role of structure, catalysis, physiological functions, pathological alterations, and methods to study TET2, with specific emphasis on epigenomics and epitranscriptomics.

Hydrogen sulphide reduces hyperhomocysteinaemia-induced endothelial ER stress by sulfhydrating protein disulphide isomerase to attenuate atherosclerosis.

Hyperhomocysteinaemia (HHcy)‐impaired endothelial dysfunction including endoplasmic reticulum (ER) stress plays a crucial role in atherogenesis. Hydrogen sulphide (H2S), a metabolic production of Hcy and gasotransmitter, exhibits preventing cardiovascular damages induced by HHcy by reducing ER stress, but the underlying mechanism is unclear. Here, we made an atherosclerosis with HHcy mice model by ApoE knockout mice and feeding Pagien diet and drinking L‐methionine water. H2S donors NaHS and GYY4137 treatment lowered plaque area and ER stress in this model. Protein disulphide isomerase (PDI), a modulation protein folding key enzyme, was up‐regulated in plaque and reduced by H2S treatment. In cultured human aortic endothelial cells, Hcy dose and time dependently elevated PDI expression, but inhibited its activity, and which were rescued by H2S. H2S and its endogenous generation key enzyme‐cystathionine γ lyase induced a new post‐translational modification‐sulfhydration of PDI. Sulfhydrated PDI enhanced its activity, and two cysteine‐terminal CXXC domain of PDI was identified by site mutation. HHcy lowered PDI sulfhydration association ER stress, and H2S rescued it but this effect was blocked by cysteine site mutation. Conclusively, we demonstrated that H2S sulfhydrated PDI and enhanced its activity, reducing HHcy‐induced endothelial ER stress to attenuate atherosclerosis development.