TET2 is required for Type I IFN-mediated inhibition of bat-origin swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered bat-origin coronavirus with fatal pathogenicity for neonatal piglets. There is no vaccine to prevent SADS-CoV infection or clinically approved drugs targeting SADS-CoV. Therefore, unraveling cellular factors that regulate SADS-CoV for cell entry is critical to understanding the viral transmission mechanism and provides a potential therapeutic target for SADS-CoV cure. Here, we showed that Type I interferon (IFN-I) pretreatment potently blocks SADS-CoV entry into cells using lentiviral pseudo-virions as targets whose entry is driven by the SADS-CoV Spike glycoprotein. IFN-I-mediated inhibition of SADS-CoV entry and replication was dramatically impaired in the absence of TET2. These results suggest TET2 is found to serve as a checkpoint of IFN-I-meditated inhibition on the cell entry of SADS-CoV, and our discovery might constitute a novel treatment option to combat against SADS-CoV.

Circular RNA 0025984 Ameliorates Ischemic Stroke Injury and Protects Astrocytes Through miR-143-3p/TET1/ORP150 Pathway.

MiR-143-3p is aberrantly expressed in patients with ischemic stroke and associated with ischemic brain injury. However, the underlying mechanisms are largely unknown. Here, we confirmed circ_0025984 and TET1 as a sponge and target of miR-143-3p, respectively, by luciferase reporter assay. In astrocytes, OGD significantly decreased circ_0025984 and TET1 levels but increased miR-143-3p levels, which was also observed in brains of mice with MCAO. Treatment with miR-143-3p inhibitor or circ_0025984 significantly decreased astrocyte apoptosis and autophagy, as well as cerebral injury and neuron loss in mice with MCAO. Notably, TET1 overexpression decreased astrocyte apoptosis and autophagy and induced promoter hypomethylation and expression of ORP150. Our results demonstrated for the first time that circ_0025984 protects astrocytes from ischemia-induced autophagy and apoptosis by targeting the miR-143-3p/TET1 pathway and might inhibit cerebral injury induced by ischemic stroke. Furthermore, our data revealed the important positive regulation of ORP150 by TET1, which could be associated with its neuroprotective role. Graphical abstract Model for signaling pathway of circ_0025984/miR-143-3p/TET1 inastrocytes cultured under OGD. In astrocytes, circ_0025984 acts as a sponge of miR-143-3p, which directly targets TET1 and decreases its expression (A). After translocatinginto the nucleus, TET1 binds to the promoter of ORP150, converts 5mC into 5hmC,leading to DNA demethylation and increased expression of ORP150 (B). In astrocytescultured under OGD, ER stress is induced and eventually leads to apoptosis andautophagy mediated by ATG7, which is regulated by circ_0025984 via ORP150 andGRP78 (C).

Muscle regeneration controlled by a designated DNA dioxygenase.

Tet dioxygenases are responsible for the active DNA demethylation. The functions of Tet proteins in muscle regeneration have not been well characterized. Here we find that Tet2, but not Tet1 and Tet3, is specifically required for muscle regeneration in vivo. Loss of Tet2 leads to severe muscle regeneration defects. Further analysis indicates that Tet2 regulates myoblast differentiation and fusion. Tet2 activates transcription of the key differentiation modulator Myogenin (MyoG) by actively demethylating its enhancer region. Re-expressing of MyoG in Tet2 KO myoblasts rescues the differentiation and fusion defects. Further mechanistic analysis reveals that Tet2 enhances MyoD binding by demethylating the flanking CpG sites of E boxes to facilitate the recruitment of active histone modifications and increase chromatin accessibility and activate its transcription. These findings shed new lights on DNA methylation and pioneer transcription factor activity regulation.

Identification and functional analysis of 9-cis-epoxy carotenoid dioxygenase (NCED) homologs in G. hirsutum.

9-cis-epoxy carotenoid dioxygenase (NCED) is a fundamental enzyme, which plays an essential role in the process of organ development and stress resistance by regulating abscisic acid (ABA) synthesis in plant. In this study, a total of 7, 7, 14 and 14 NCED genes were identified from the genomes of G. arboreum, G. raimondii, G. barbadense and G. hirsutum, respectively. Phylogenetic tree showed that all forty-two NCED genes could be classified into three groups in cotton genus. Collinear analysis revealed that the NCED genes in G. hirsutum were not amplified by tandem repeats after polyploidy events. The function of NCED genes was evaluated between two accessions with contrasting plant height. The results showed that expression of the NCED genes in dwarf accession was higher than that in taller ones. GhNCED1-silenced cotton plants confirmed that suppression of NCED genes could increase the plant height, but reduce the resistance abilities to drought and salt stress. Our study systematically identified the homologs of NCED genes and their functions in cotton, which could provide new genetic resources for improving plant height and stress in future cotton breeding.

Double NCED isozymes control ABA biosynthesis for ripening and senescent regulation in peach fruits.

During ripening, peach fruits (Prunus persica L. Batsch) rapidly progress to the senescent stage, resulting in a brief shelf life. Abscisic acid (ABA) plays an important role in regulating the ripening process, both in climacteric and non-climacteric fruits. A key enzyme for ABA biosynthesis in higher plants is 9-cis-epoxycarotenoid dioxygenase (NCED). In this study, two NCED isozymes, PpNCED1 and PpNCED5, were identified in peach fruits. While both NCED genes had similar transcriptional patterns (up-regulation) at the beginning of peach ripening, PpNCED5 showed a consistently lower expression level than PpNCED1. During the post-harvest stage, gene expression of PpNCED1 declined, while PpNCED5 expression increased relative to PpNCED1 expression. Considering the dynamic process of ABA accumulation during fruit ripening and senescence in peach, this study indicates that both NCED genes cooperatively control ABA biosynthesis in peach fruits. Moreover, spatio-temporal expression and transcriptional response to hormone and abiotic stress suggested that there is functional divergence between PpNCED1 and PpNCED5 genes in peach. A carotenoid-rich callus system was used to verify the function of PpNCED1 and PpNCED5. In the transgenic callus system, both PpNCED1 and PpNCED5 isozymes promoted ABA biosynthesis, which likely accelerated cell senescence through activating ROS signals. The results from this study provide evidence supporting an ABA biosynthetic regulation process via the two NCED genes in peach fruit, and suggest a mechanism of ABA-induced fruit ripening and senescence.

2-Oxoglutarate-dependent dioxygenases in cancer.

2-Oxoglutarate-dependent dioxygenases (2OGDDs) are a superfamily of enzymes that play diverse roles in many biological processes, including regulation of hypoxia-inducible factor-mediated adaptation to hypoxia, extracellular matrix formation, epigenetic regulation of gene transcription and the reprogramming of cellular metabolism. 2OGDDs all require oxygen, reduced iron and 2-oxoglutarate (also known as α-ketoglutarate) to function, although their affinities for each of these co-substrates, and hence their sensitivity to depletion of specific co-substrates, varies widely. Numerous 2OGDDs are recurrently dysregulated in cancer. Moreover, cancer-specific metabolic changes, such as those that occur subsequent to mutations in the genes encoding succinate dehydrogenase, fumarate hydratase or isocitrate dehydrogenase, can dysregulate specific 2OGDDs. This latter observation suggests that the role of 2OGDDs in cancer extends beyond cancers that harbour mutations in the genes encoding members of the 2OGDD superfamily. Herein, we review the regulation of 2OGDDs in normal cells and how that regulation is corrupted in cancer.

Dietary ß-Cryptoxanthin Inhibits High-Refined Carbohydrate Diet-Induced Fatty Liver via Differential Protective Mechanisms Depending on Carotenoid Cleavage Enzymes in Male Mice.

BACKGROUND: ß-Cryptoxanthin (BCX), a provitamin A carotenoid shown to protect against nonalcoholic fatty liver disease (NAFLD), can be cleaved by ß-carotene-15,15'-oxygenase (BCO1) to generate vitamin A, and by ß-carotene-9',10'-oxygenase (BCO2) to produce bioactive apo-carotenoids. BCO1/BCO2 polymorphisms have been associated with variations in plasma carotenoid amounts in both humans and animals. OBJECTIVES: We investigated whether BCX feeding inhibits high refined-carbohydrate diet (HRCD)-induced NAFLD, dependent or independent of BCO1/BCO2. METHODS: Six-week-old male wild-type (WT) and BCO1-/-/BCO2-/- double knockout (DKO) mice were randomly fed HRCD (66.5% of energy from carbohydrate) with or without BCX (10 mg/kg diet) for 24 wk. Pathological and biochemical variables were analyzed in the liver and mesenteric adipose tissues (MATs). Data were analyzed by 2-factor ANOVA. RESULTS: Compared to their respective HRCD controls, BCX reduced hepatic steatosis severity by 33‒43% and hepatic total cholesterol by 43‒70% in both WT and DKO mice (P < 0.01). Hepatic concentrations of BCX, but not retinol and retinyl palmitate, were 33-fold higher in DKO mice than in WT mice (P < 0.001). BCX feeding increased the hepatic fatty acid oxidation protein peroxisome proliferator-activated receptor-α, and the cholesterol efflux gene ATP-binding cassette transporter5, and suppressed the lipogenesis gene acetyl-CoA carboxylase 1 (Acc1) in the MAT of WT mice but not DKO mice (P < 0.05). BCX feeding decreased the hepatic lipogenesis proteins ACC and stearoyl-CoA desaturase-1 (3-fold and 5-fold) and the cholesterol synthesis genes 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and HMG-CoA synthase 1 (2.7-fold and 1.8-fold) and increased the cholesterol catabolism gene cholesterol 7α-hydroxylase (1.9-fold) in the DKO but not WT mice (P < 0.05). BCX feeding increased hepatic protein sirtuin1 (2.5-fold) and AMP-activated protein kinase (9-fold) and decreased hepatic farnesoid X receptor protein (80%) and the inflammatory cytokine gene Il6 (6-fold) in the MAT of DKO mice but not WT mice (P < 0.05). CONCLUSION: BCX feeding mitigates HRCD-induced NAFLD in both WT and DKO mice through different mechanisms in the liver-MAT axis, depending on the presence or absence of BCO1/BCO2.

TET enzymes in neurophysiology and brain function.

The dynamic nature of epigenetic DNA modifications is crucial for regulating gene expression in an experience-dependent manner and, thus, a potential mediator of neuronal plasticity and behavior. The discovery of the involvement of 5-hydroxymethylcytosine (5hmC) and Ten Eleven Translocation (TET) family of enzymes in the demethylation pathway uncovered a potential link between neuronal TET protein function and cognitive processes. In this review, we provide an overview on how profile of 5hmC and TET enzymes are powerful mechanisms to explain neuronal plasticity and long-term behaviors, such as cognition. More specifically, we discuss how the current knowledge integrates the function of each TET enzyme in neurophysiology and brain function.

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kß,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

OsARD4 encoding an acireductone dioxygenase improves root architecture in rice by promoting development of secondary roots.

This study was aimed at unravelling the molecular basis of root growth behavior in a drought-tolerant upland rice genotype, Nootripathu. Root tips of Nootripathu were found to possess shorter root caps and a greater number of dividing cells, favoring faster elongation compared to shallow-rooted IR20. Width and length of cortical cells in the roots of rapidly growing Nootripathu were found to be two to three times higher than IR20. Evaluation of shallow-rooted IR20, deep-rooted Nootripathu and their Recombinant Inbred Lines (RILs) for root characteristics revealed the presence of genetic variation for root traits among RILs. 2D-PAGE analysis of proteins in roots of IR20, Nootripathu and bulks of extreme RILs differing in root traits resulted in the identification of proteins co-segregating with root growth behavior and co-localized with QTLs for root traits. A putative candidate gene, OsARD4, encoding an "acireductone dioxygenase" was validated for its role in modulating the root growth pattern through genetic transformation. Transgenic ASD16 rice plants engineered for the overexpression of OsARD4 exhibited root growth characteristics similar to those of Nootripathu, including faster radical emergence, more rapid elongation of primary roots, early initiation of crown/lateral roots, and higher root biomass than the non-transgenic plants.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Research progress on 5hmC and TET dioxygenases in neurodevelopment and neurological diseases.

The development of the nervous system is coordinately regulated by multiple interacting factors. If a certain factor is altered or mutated, the coordinated developmental processes could be disrupted, resulting in neurological diseases. The 5-hydroxymethylcytosine (5hmC) is an intermediate product of the DNA demethylation processes. 5hmC and its metabolic enzymes, the ten-eleven translocation protein-TET family of dioxygenases, have recently been identified as new epigenetic players important in the regulation of the nervous system development, as well as in cognition, memory and other neurological functions. In various studies on neurodevelopment and neurodegeneration related diseases, the levels of 5hmC and TET proteins could be differentially regulated during development and/or disease pathogenesis, suggesting the potentially critical roles of 5hmC and TETs in these neural developmental and disease processes. In this review, we summarize the recent advances in research on 5hmC and TET dioxygenases in the regulation of neurodevelopment and neurological diseases, thereby providing significant insights on the involvements of 5hmC and TETs in neurodevelopment and on establishing new therapeutic strategies for human neurological diseases.

Targeted Metabolomics Reveals Abnormal Hepatic Energy Metabolism by Depletion of ß-Carotene Oxygenase 2 in Mice.

ß-carotene oxygenase 2 (BCO2) is a carotenoid cleavage enzyme located in the inner mitochondrial membrane. Ablation of BCO2 impairs mitochondrial function leading to oxidative stress. Herein, we performed a targeted metabolomics study using ultrahigh performance liquid chromatography-tandem mass spectroscopy and gas chromatography-mass spectroscopy to discriminate global metabolites profiles in liver samples from six-week-old male BCO2 systemic knockout (KO), heterozygous (Het), and wild type (WT) mice fed a chow diet. Principal components analysis revealed distinct differences in metabolites in the livers of KO mice, compared to WT and Het mice. However, no marked difference was found in the metabolites of the Het mouse liver compared to the WT. We then conducted random forest analysis to classify the potential biomarkers to further elucidate the different metabolomics profiles. We found that systemic ablation of BCO2 led to perturbations in mitochondrial function and metabolism in the TCA cycle, amino acids, carnitine, lipids, and bile acids. In conclusion, BCO2 is essential to macronutrient and mitochondrial metabolism in the livers of mice. The ablation of BCO2 causes dysfunctional mitochondria and altered energy metabolism, which further leads to systemic oxidative stress and inflammation. A single functional copy of BCO2 largely rescues the hepatic metabolic homeostasis in mice.

Enzymatic study on AtCCD4 and AtCCD7 and their potential to form acyclic regulatory metabolites.

The Arabidopsis carotenoid cleavage dioxygenase 4 (AtCCD4) is a negative regulator of the carotenoid content of seeds and has recently been suggested as a candidate for the generation of retrograde signals that are thought to derive from the cleavage of poly-cis-configured carotene desaturation intermediates. In this work, we investigated the activity of AtCCD4 in vitro and used dynamic modeling to determine its substrate preference. Our results document strict regional specificity for cleavage at the C9-C10 double bond in carotenoids and apocarotenoids, with preference for carotenoid substrates and an obstructing effect on hydroxyl functions, and demonstrate the specificity for all-trans-configured carotenes and xanthophylls. AtCCD4 cleaved substrates with at least one ionone ring and did not convert acyclic carotene desaturation intermediates, independent of their isomeric states. These results do not support a direct involvement of AtCCD4 in generating the supposed regulatory metabolites. In contrast, the strigolactone biosynthetic enzyme AtCCD7 converted 9-cis-configured acyclic carotenes, such as 9-cis-ζ-carotene, 9'-cis-neurosporene, and 9-cis-lycopene, yielding 9-cis-configured products and indicating that AtCCD7, rather than AtCCD4, is the candidate for forming acyclic retrograde signals.

Molecular aspects of ß, ß-carotene-9', 10'-oxygenase 2 in carotenoid metabolism and diseases.

Carotenoids, the carotenes and xanthophylls, are essential components in human nutrition. ß, ß-carotene-9', 10'-oxygenase 2 (BCO2), also named as ß, ß-carotene-9', 10'-dioxygenase 2 (BCDO2) catalyzes the asymmetrical cleavage of carotenoids, whereas ß, ß-carotene-15, 15'-monooxygenase (BCMO1) conducts the symmetrical cleavage of pro-vitamin A carotenoids into retinoid. Unlike BCMO1, BCO2 has a broader substrate specificity and has been considered an alternative way to produce vitamin A. In contrast to BCMO1, a cytoplasmic protein, BCO2 is located in the inner mitochondrial membrane. The difference in cellular compartmentalization may reflect the different substrate specificity and physiological functions with respect to BCMO1 and BCO2. The BCO2 gene mutations are proven to be associated with yellow color of skin and fat tissue and milk in livestock. Mutation in intron 2 of BCO2 gene is also supposed to be related to the expression of IL-18, a pro-inflammatory cytokine associated with obesity, cardiovascular diseases, and type 2 diabetes. Further, BCO2 is associated with the development of mitochondrial oxidative stress, macular degeneration, anemia, and hepatic steatosis. This review of the literature will mostly address recent updates regarding the role of BCO2 in carotenoid metabolism, and discuss the potential impacts of BCO2 protein and the mutations in mammalian diseases.

Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron.

The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid ß-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma.

ZmCCD7/ZpCCD7 encodes a carotenoid cleavage dioxygenase mediating shoot branching.

MAIN CONCLUSION: ZmCCD7/ZpCCD7 encodes a carotenoid cleavage dioxygenase that may mediate strigolactone biosynthesis highly responsive to phosphorus deficiency and undergoes negative selection over domestication from Zea ssp. parviglumis to Zea mays. Carotenoid cleavage dioxygenase 7 (CCD7) functions to suppress shoot branching by controlling strigolactone biosynthesis. However, little is known about CCD7 and its functions in maize and its ancestor (Zea ssp. parviglumis) with numerous shoot branches. We found that ZmCCD7 and ZpCCD7 had the same coding sequence, indicating negative selection of the CCD7 gene over domestication from Zea ssp. parviglumis to Zea mays. CCD7 expression was highly responsive to phosphorus deficiency in both species, especially in the meristematic zone and the pericycle of the elongation zone of maize roots. Notably, the crown root had the strongest ZmCCD7 expression in the meristematic zone under phosphorus limitation. Transient expression of GFP tagged ZmCCD7/ZpCCD7 in maize protoplasts indicated their localization in the plastid. Further, ZmCCD7/ZpCCD7 efficiently catalyzed metabolism of six different linear and cyclic carotenoids in E. coli, and generated ß-ionone by cleaving ß-carotene at the 9,10 (9',10') position. Together with suppression of shoot branching in the max3 mutant by transformation of ZmCCD7/ZpCCD7, our work suggested that ZmCCD7/ZpCCD7 encodes a carotenoid cleavage dioxygenase mediating strigolactone biosynthesis in maize and its ancestor.

Metal-Dependent Function of a Mammalian Acireductone Dioxygenase.

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe(2+) or Ni(2+)) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate the metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, which is the penultimate step in methionine salvage. The nickel-bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO), and 3-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe(2+)-bound protein, which shows about 10-fold higher activity than that of others, catalyzes on-pathway chemistry, whereas the Ni(2+), Co(2+), or Mn(2+) forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by the metal ion identity, with Ni(2+)-bound MmARD being the most stable, followed by Co(2+) and Fe(2+), and Mn(2+)-bound ARD being the least stable. Ni(2+)- and Co(2+)-bound MmARD were crystallized, and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination, and the catalytic mechanism.

Hypoxia Drives Breast Tumor Malignancy through a TET-TNFα-p38-MAPK Signaling Axis.

Hypoxia is a hallmark of solid tumors that drives malignant progression by altering epigenetic controls. In breast tumors, aberrant DNA methylation is a prevalent epigenetic feature associated with increased risk of metastasis and poor prognosis. However, the mechanism by which hypoxia alters DNA methylation or other epigenetic controls that promote breast malignancy remains poorly understood. We discovered that hypoxia deregulates TET1 and TET3, the enzymes that catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby leading to breast tumor-initiating cell (BTIC) properties. TET1/3 and 5hmC levels were closely associated with tumor hypoxia, tumor malignancy, and poor prognosis in breast cancer patients. Mechanistic investigations showed that hypoxia leads to genome-wide changes in DNA hydroxymethylation associated with upregulation of TNFα expression and activation of its downstream p38-MAPK effector pathway. Coordinate functions of TET1 and TET3 were also required to activate TNFα-p38-MAPK signaling as a response to hypoxia. Our results reveal how signal transduction through the TET-TNFα-p38-MAPK signaling axis is required for the acquisition of BTIC characteristics and tumorigenicity in vitro and in vivo, with potential implications for how to eradicate BTIC as a therapeutic strategy.

TET Family of Dioxygenases: Crucial Roles and Underlying Mechanisms.

DNA methylation plays an important role in the epigenetic regulation of mammalian gene expression. TET (ten-eleven translocation) proteins, newly discovered demethylases, have sparked great interest since their discovery. TET proteins catalyze 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in 3 consecutive Fe(II)- and 2-oxoglutarate (2-OG)-dependent oxidation reactions. TET proteins dynamically regulate global or locus-specific 5-methylcytosine and/or 5-hydroxymethylcytosine levels by facilitating active DNA demethylation. In fact, in addition to their role as methylcytosine dioxygenases, TET proteins are closely related to histone modification, interact with metabolic enzymes as well as other proteins, and cooperate in transcriptional regulation. In this review, we summarize the recent progress in this exciting field, highlighting the molecular mechanism by which TET enzymes regulate gene expression and their functions in health and disease. We also discuss the therapeutic potential of targeting TET proteins and aberrant DNA modifications.