Dioxygenase Family Research Articles

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

Comparative analysis of Tet2 catalytic-deficient and knockout bone marrow over time

TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.

Single-cell Transcriptome Landscape of DNA Methylome Regulators Associated with Orofacial Clefts in the Mouse Dental Pulp.

Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. The biomedical research institution. This study did not include patients. This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes.

Computational Study of the Fe(II) and α-Ketoglutarate-Dependent Aryloxyalkanoate Dioxygenase (AAD-1) in the Degradation of the Herbicide 2,4-Dichlorophenoxyacetic Acid.

The AAD-1 enzyme belongs to the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent nonheme aryloxyalkanoate dioxygenase family (AADs), which catalyzes the breakdown of 2,4-dichlorophenoxyacetic acid (2,4-D, an active ingredient of thousands of commercial herbicides) by using the highly active Fe(IV)═O complex. Multiple species of bacteria degrade 2,4-D via a pathway initiated by AADs; however, the detail of how they promote the cleavage of the ether C-O bond to generate 2,4-dichlorophenol (2,4-DCP) and glyoxylate is still unclear, which is the prerequisite for the further degradation of these halogenated aromatics. In this work, based on the crystal structure of AAD-1, the computational models were constructed, and a series of QM/MM and QM-only calculations were performed to explore the cleavage of the ether bond in 2,4-D with the catalysis of AAD-1. Our calculations reveal that AAD-1 may be only responsible for the hydroxylation of the substrate to generate the intermediate hemiacetal, which corresponds to an overall energy barrier of 14.2 kcal/mol on the quintet state surface, and the decomposition of the hemiacetal in the active site center of AAD-1 was calculated to be rather slow, corresponding to an energy barrier of 24.5 kcal/mol. In contrast, the decomposition of the free hemiacetal molecule in a solvent was calculated to be quite easy. Whether the decomposition of the hemiacetal occurs inside or outside the activation site is still worthy of experimental verification.

OGFOD1 modulates the transcriptional and proteomic landscapes to alter isoproterenol-induced hypertrophy susceptibility

Cardiac hypertrophy is associated with increased translation. However, little is known of the mechanisms that regulate translation in hypertrophy. Members of the 2-oxoglutarate-dependent dioxygenase family regulate several aspects of gene expression, including translation. An important member of this family is OGFOD1. Here, we show OGFOD1 accumulates in failing human hearts. Upon OGFOD1 deletion, murine hearts showed transcriptomic and proteomic changes, with only 21 proteins and mRNAs (0.6%) changing in the same direction. Additionally, OGFOD1-KO mice were protected from induced hypertrophy, supporting a role for OGFOD1 in the cardiac response to chronic stress.

Abstract 2226: Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression

Abstract Loss of function in DNA methylation regulators such as the Ten-eleven translocation (TET1-3) proteins, have been implicated in the progression of lymphoma. TET enzymes constitute a family of iron and αKG-dependent dioxygenases that oxidize 5-methylcytosine in DNA to promote demethylation and the reversal of gene silencing. The TET enzymes require vitamin C for their optimal catalytic activity, however, many patients with hematological malignancies are vitamin C deficient. Enhancing and restoring TET function with high-dose vitamin C has been shown to block the progression of solid tumors and blood malignancies. A combination of genetic and epigenetic diversity in lymphoma can result in the aberrant overexpression of oncogenes, loss of tumor suppressor function, and genomic instability. Our research models Bcl2-driven B cell lymphoma with Tet-deficiency using transgenic RNAi for the knockdown of Tet1 or Tet2. In vitro, our results have shown that this model shows increased self-renewal capacity of early B cell progenitors and precursors that can be suppressed by genetic restoration of Tet expression or upon treatment with vitamin C. In vivo, the B cell compartment of mice with Bcl2 overexpression and Tet1 or Tet2 deficiency exhibit a block in B cell maturation during a long period of pre-malignancy, and a decreased frequency of follicular B cells in the spleen from 3 months of age. Our aim is to determine mechanistically the effects of Tet-deficiency on B cell lymphoma progression and whether vitamin C can restore Tet activity to suppress lymphoma progression. Citation Format: Minh Quang Lam, Luisa Cimmino. Modeling the role of Tet-deficiency and restoration in Bcl2-driven B cell lymphoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2226.

A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (CmCGG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H2O2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening.

Abstract 2600: BBOX1 is a novel tumor suppressor in kidney cancer

Abstract Renal cell carcinoma (RCC) is a common type of cancer with a steady increase in new cases and deaths for the past several decades while its development is not fully understood. We previously identified the γ-butyrobetaine hydroxylase 1 (BBOX1), a member of the 2-oxoglutarate dependent dioxygenase family whereas functionally uncharacterized previously in human cancers, plays a vital oncogenic role in triple-negative breast cancer. However, the role of BBOX1 in other cancers such as kidney cancer remains unclear. We found that BBOX1 is a highly context-dependent enzyme, reflected by its expression only detected in a few organs such as kidney. Intriguingly, according to our immunohistochemistry staining results and the data from The Human Protein Atlas database, BBOX1 is only expressed in the proximal tubule cells where clear cell RCC (ccRCC, the most common form of kidney cancer) derives from, indicating a connection of BBOX1 with kidney malignant transformation. In this study, we investigated the BBOX1 mRNA and protein expression in paired normal and tumor ccRCC patient samples from the UTSW Kidney Cancer Specialized Program of Research Excellence (SPORE) program, as well as patient data from The Cancer Genome Atlas (TCGA) or Clinical Proteomic Tumor Analysis Consortium (CPTAC), which consistently showed that BBOX1 is remarkably downregulated in the ccRCC patient tumors compared to normal kidneys, and its low expression associates with worse prognosis in ccRCC patients. Furthermore, expression of BBOX1 was highly presented in the human primary renal proximal tubule epithelial cells (RPTEC) while robustly suppressed after malignant transformation of the RPTEC cells. These data suggest a pathological loss of BBOX1 during RCC progression. Restoration of BBOX1 in multiple human ccRCC cell lines suppressed xenograft tumor growth in mice but not in the 2D in vitro growth with normal tissue culture conditions, suggesting BBOX1 plays an important role in ccRCC tumor development. By comparing the gene expression profiles from transcriptomics analyses in the control or BBOX1 overexpression samples either from xenograft tumors or cells grown in normal 2D culture, we found that BBOX1 suppresses the mTORC1 signaling only in the in vivo condition. Interestingly, we could recapitulate this phenomenon by culturing the control or BBOX1 overexpression ccRCC cells in a physiologic medium but not the standard cell culture medium. These data suggested BBOX1 may regulate the mTORC1 signaling depending on the nutrient status of the tumor cells. Overall, we identified BBOX1 as a novel tumor suppressor in ccRCC. Citation Format: Chengheng Liao, Liwei Jia, Hua Zhong, Lianxin Hu, Payal Kapur, James Brugarolas, Qing Zhang. BBOX1 is a novel tumor suppressor in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2600.

The crosstalk between the RNA demethylase, non‐coding RNAs, and transcription factors in gastric cancer: An ALKBH5 perspective

The crosstalk between the RNA demethylase, non‐coding RNAs, and transcription factors in gastric cancer: An ALKBH5 perspective

Genome-Wide Identification of 2-Oxoglutarate and Fe (II)-Dependent Dioxygenase (2ODD-C) Family Genes and Expression Profiles under Different Abiotic Stresses in Camellia sinensis (L.)

The 2-oxoglutarate and Fe (II)-dependent dioxygenase (2ODD-C) family of 2-oxoglutarate-dependent dioxygenases potentially participates in the biosynthesis of various metabolites under various abiotic stresses. However, there is scarce information on the expression profiles and roles of 2ODD-C genes in Camellia sinensis. We identified 153 Cs2ODD-C genes from C. sinensis, and they were distributed unevenly on 15 chromosomes. According to the phylogenetic tree topology, these genes were divided into 21 groups distinguished by conserved motifs and an intron/exon structure. Gene-duplication analyses revealed that 75 Cs2ODD-C genes were expanded and retained after WGD/segmental and tandem duplications. The expression profiles of Cs2ODD-C genes were explored under methyl jasmonate (MeJA), polyethylene glycol (PEG), and salt (NaCl) stress treatments. The expression analysis showed that 14, 13, and 49 Cs2ODD-C genes displayed the same expression pattern under MeJA and PEG treatments, MeJA and NaCl treatments, and PEG and NaCl treatments, respectively. A further analysis showed that two genes, Cs2ODD-C36 and Cs2ODD-C21, were significantly upregulated and downregulated after MeJA, PEG, and NaCl treatments, indicating that these two genes played positive and negative roles in enhancing the multi-stress tolerance. These results provide candidate genes for the use of genetic engineering technology to modify plants by enhancing multi-stress tolerance to promote phytoremediation efficiency.

R97 at "Handlebar" Binding Mode in Active Pocket Plays an Important Role in Fe(II)/α-Ketoglutaric Acid-Dependent Dioxygenase cis-P3H-Mediated Selective Synthesis of (2S,3R)-3-Hydroxypipecolic Acid.

Pipecolic acid (Pip) and its derivative hydroxypipecolic acids, such as (2S,3R)-3-hydroxypipecolic acid (cis-3-L-HyPip), are components of many natural and synthetic bioactive molecules. Fe(II)/α-ketoglutaric acid (Fe(II)/2-OG)-dependent dioxygenases can catalyze the hydroxylation of pipecolic acid. However, the available enzymes with desired activity and selectivity are limited. Herein, we compare the possible candidates in the Fe(II)/2-OG-dependent dioxygenase family, and cis-P3H is selected for potentially catalyzing selective hydroxylation of L-Pip. cis-P3H was further engineered to increase its catalytic efficiency toward L-Pip. By analyzing the structural confirmation and residue composition in substrate-binding pocket, a "handlebar" mode of molecular interactions is proposed. Using molecular docking, virtual mutation analysis, and dynamic simulations, R97, E112, L57, and G282 were identified as the key residues for subsequent site-directed saturation mutagenesis of cis-P3H. Consequently, the variant R97M showed an increased catalytic efficiency toward L-Pip. In this study, the kcat/Km value of the positive mutant R97M was about 1.83-fold that of the wild type. The mutation R97M would break the salt bridge between R97 and L-Pip and weaken the positive-positive interaction between R97 and R95. Therefore, the force on the amino and carboxyl groups of L-Pip was lightly balanced, allowing the molecule to be stabilized in the active pocket. These results provide a potential way of improving cis-P3H catalytic activity through rational protein engineering.

Molecular Characterization of a Stereoselective and Promiscuous Flavanone 3-Hydroxylase fromCarthamus tinctoriusL

Flavanone 3-hydroxylases (F3Hs) belong to the 2-oxoglutarate-dependent dioxygenase family and play an important role in plant flavonoid biosynthesis. However, the stereoselective catalytic mechanism and substrate promiscuity of this type of enzyme are not well understood. In this study, we identified and biochemically characterized CtF3H1, an F3H from Carthamus tinctorius, a plant used in traditional Chinese medicine that exhibits high stereoselectivity and substrate promiscuity toward structurally diverse (2S)-flavanones. Isothermal titration calorimetry revealed that CtF3H1 exhibits distinctly different binding behaviors with (2S)-flavanone (2S-naringenin) and (2R)-flavanone (2R-naringenin), and these differences govern its stereoselectivity. An investigation of the structure-activity relationships between the enzyme and its substrates demonstrated that 7-OH and/or 4'-OH are necessary for regio- and stereoselective 3-hydroxylation of (2S)-flavanones. Homology modeling and molecular docking combined with site-directed mutagenesis identified the amino acid residues necessary for hydroxylation. These findings demonstrate the potential versatility of CtF3H1 in regio- and stereohydroxylation and provide molecular insights into the catalytic mechanism of F3H for further enzyme engineering.

Tet1 deficiency exacerbates oxidative stress in acute kidney injury by regulating superoxide dismutase.

Rationale: Increased methylation of key genes has been observed in kidney diseases, suggesting that the ten-eleven translocation (Tet) methyl-cytosine dioxygenase family as well as 5mC oxidation may play important roles. As a member of the Tet family, the role of Tet1 in acute kidney injury (AKI) remains unclear. Methods: Tet1 knockout mice, with or without tempol treatment, a scavenger of reactive oxygen species (ROS), were challenged with ischemia and reperfusion (I/R) injury or unilateral ureteral obstruction (UUO) injury. RNA-sequencing, Western blotting, qRT-PCR, bisulfite sequencing, chromatin immunoprecipitation, immunohistochemical staining, and dot blot assays were performed. Results: Tet1 expression was rapidly upregulated following I/R or UUO injury. Moreover, Tet1 knockout mice showed increased renal injury and renal cell death, increased ROS accumulation, G2/M cell cycle arrest, inflammation, and fibrosis. Severe renal damage in injured Tet1 knockout mice was alleviated by tempol treatment. Mechanistically, Tet1 reduced the 5mC levels in an enzymatic activity-dependent manner on the promoters of Sod1 and Sod2 to promote their expression, thus lowering injury-induced excessive ROS and reducing I/R or UUO injury. Conclusions: Tet1 plays an important role in the development of AKI by promoting SOD expression through a DNA demethylase-dependent mechanism.

ABA biosynthesis gene OsNCED3 contributes to preharvest sprouting resistance and grain development in rice.

Preharvest sprouting (PHS) is an unfavorable trait in cereal crops and causes serious yield loss. However, the molecular mechanism underlying PHS remains largely elusive. Here, we identified a member of 9-cis-epoxycarotenoid dioxygenase family, OsNCED3, which regulates PHS and grain development in rice (Oryza sativa L.). OsNCED3 encodes a chloroplast-localized abscisic acid(ABA) biosynthetic enzyme highly expressed in the embryo of developing seeds. Disruption of OsNCED3 by CRISPR/Cas9-mediated mutagenesis led to a lower ABA and higher gibberellic acid(GA) levels (thus a skewed ABA/GA ratio) in the embryo, promoting embryos growth and breaking seed dormancy before seed maturity and harvest, thus decreased seed dormancy and enhanced PHS in rice. However, the overexpression of OsNCED3 enhanced PHS resistance by regulating proper ABA/GA ratio in the embryo. Intriguingly, the overexpression of OsNCED3 resulted in increased grain size and weight, whereas the disruption of OsNCED3 function decreased grain size and weight. Nucleotide diversity analyses suggested that OsNCED3 may be selected during japonica populations adaptation of seed dormancy and germination. Taken together, we have identified a new OsNCED regulator involved rice PHS and grain development, and provide a potential target gene for improving PHS resistance and grain development in rice.

TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons.

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide–expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.

DNA methylation reprogramming of genomic imprints in the mammalian germline: A TET-centric view.

The mammalian genome undergoes extensive epigenetic reprogramming twice during development, once during gestation when primordial germ cells (PGCs) are specified from somatic cells and a second time after fertilization in the preimplantation embryo. PGC differentiation into germ cells involves DNA demethylation and subsequent remethylation. DNA demethylation takes place in two waves in the mouse germline, an early phase where most of the genome is demethylated by replication coupled passive demethylation, and a second phase predominated by active DNA demethylation. Imprinted genes, CpG islands on the inactive X chromosome of females, and germline-specific genes are among those loci that are demethylated late. The Ten-Eleven Translocation (TET) family of 5mC dioxygenases has emerged as active demethylating enzymes that are critical to achieving a DNA hypomethylated state, with TET1 being the most important for imprinted genes. Here, we discuss DNA methylation dynamics in the mammalian genome, with a particular emphasis on DNA demethylation in the germline and the requirement for TET1 in imprinted gene reprogramming.

TET enzymes regulate skeletal development through increasing chromatin accessibility of RUNX2 target genes

The Ten-eleven translocation (TET) family of dioxygenases mediate cytosine demethylation by catalyzing the oxidation of 5-methylcytosine (5mC). TET-mediated DNA demethylation controls the proper differentiation of embryonic stem cells and TET members display functional redundancy during early gastrulation. However, it is unclear if TET proteins have functional significance in mammalian skeletal development. Here, we report that Tet genes deficiency in mesoderm mesenchymal stem cells results in severe defects of bone development. The existence of any single Tet gene allele can support early bone formation, suggesting a functional redundancy of TET proteins. Integrative analyses of RNA-seq, Whole Genome Bisulfite Sequencing (WGBS), 5hmC-Seal and Assay for Transposase-Accessible Chromatin (ATAC-seq) demonstrate that TET-mediated demethylation increases the chromatin accessibility of target genes by RUNX2 and facilities RUNX2-regulated transcription. In addition, TET proteins interact with RUNX2 through their catalytic domain to regulate cytosine methylation around RUNX2 binding region. The catalytic domain is indispensable for TET enzymes to regulate RUNX2 transcription activity on its target genes and to regulate bone development. These results demonstrate that TET enzymes function to regulate RUNX2 activity and maintain skeletal homeostasis.

A Moss 2-Oxoglutarate/Fe(II)-Dependent Dioxygenases (2-ODD) Gene of Flavonoids Biosynthesis Positively Regulates Plants Abiotic Stress Tolerance.

Flavonoids, the largest group of polyphenolic secondary metabolites present in all land plants, play essential roles in many biological processes and defense against abiotic stresses. In the flavonoid biosynthesis pathway, flavones synthase I (FNSI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and anthocyanidin synthase (ANS) all belong to 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs) family, which catalyzes the critical oxidative reactions to form different flavonoid subgroups. Here, a novel 2-ODD gene was cloned from Antarctic moss Pohlia nutans (Pn2-ODD1) and its functions were investigated both in two model plants, Physcomitrella patens and Arabidopsis thaliana. Heterologous expression of Pn2-ODD1 increased the accumulation of anthocyanins and flavonol in Arabidopsis. Meanwhile, the transgenic P. patens and Arabidopsis with expressing Pn2-ODD1 exhibited enhanced tolerance to salinity and drought stresses, with larger gametophyte sizes, better seed germination, and longer root growth. Heterologous expression of Pn2-ODD1 in Arabidopsis also conferred the tolerance to UV-B radiation and oxidative stress by increasing antioxidant capacity. Therefore, we showed that Pn2-ODD1 participated in the accumulation of anthocyanins and flavonol in transgenic plants, and regulated the tolerance to abiotic stresses in plants, contributing to the adaptation of P. nutans to the polar environment.

Mechanisms that regulate the activities of TET proteins.

The ten-eleven translocation (TET) family of dioxygenases consists of three members, TET1, TET2, and TET3. All three TET enzymes have Fe+2 and α-ketoglutarate (α-KG)-dependent dioxygenase activities, catalyzing the 1st step of DNA demethylation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Gene knockout studies demonstrated that all three TET proteins are involved in the regulation of fetal organ generation during embryonic development and normal tissue generation postnatally. TET proteins play such roles by regulating the expression of key differentiation and fate-determining genes via (1) enzymatic activity-dependent DNA methylation of the promoters and enhancers of target genes; and (2) enzymatic activity-independent regulation of histone modification. Interacting partner proteins and post-translational regulatory mechanisms regulate the activities of TET proteins. Mutations and dysregulation of TET proteins are involved in the pathogenesis of human diseases, specifically cancers. Here, we summarize the research on the interaction partners and post-translational modifications of TET proteins. We also discuss the molecular mechanisms by which these partner proteins and modifications regulate TET functioning and target gene expression. Such information will help in the design of medications useful for targeted therapy of TET-mutant-related diseases.

Lack of Major Genome-Wide DNA Methylation Changes in Succinate-Treated Human Epithelial Cells

The tricarboxylic acid (TCA) metabolite, succinate, is a competitive inhibitor of dioxygenase enzymes that require alpha ketoglutarate as a cofactor. One family of dioxygenases are the ten-eleven translocation (TET) proteins, which oxidize 5-methylcytosine to promote DNA demethylation. Inhibition of DNA demethylation is expected to lead to DNA hypermethylation, at least at genomic regions at which TET proteins are engaged. We treated human bronchial epithelial cells with succinate for five days and confirmed its effect on TET protein function by observing diminished formation of 5-hydroxymethylcytosine, the first oxidation product of the TET enzymatic reaction. We then analyzed global DNA methylation patterns by performing whole-genome bisulfite sequencing. Unexpectedly, we did not observe differentially methylated regions (DMRs) that reached genome-wide statistical significance. We observed a few regions of clustered DNA hypomethylation, which was also not expected based on the proposed mechanisms. We discuss potential explanations for our observations and the implications of these findings for tumorigenesis.