Ruichuan Yin

Ascorbic Acid Enhances Tet-Mediated 5-Methylcytosine Oxidation and Promotes DNA Demethylation in Mammals

DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.

N6-Methyladenine DNA Modification in Drosophila

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Detection of Human Urinary 5-Hydroxymethylcytosine by Stable Isotope Dilution HPLC-MS/MS Analysis

The sixth DNA base 5-hydroxymethylcytosine (5hmC) is the major oxidation product of the epigenetic modification 5-methylcytosine (5mC), mediating DNA demethylation in mammals. Reduced 5hmC levels are found to be linked with various tumors and neurological diseases; therefore, 5hmC is an emerging biomarker for disease diagnosis, treatment, and prognosis. Due to its advantages of being sterile, easily accessible in large volumes, and noninvasive to patients, urine is a favored diagnostic biofluid for 5hmC analysis. Here we developed an accurate, sensitive, and specific assay for quantification of 5mC, 5hmC, and other DNA demethylation intermediates in human urine. The urinary samples were desalted and enriched using off-line solid-phase extraction, followed by stable isotope dilution HPLC-MS/MS analysis for 5hmC and 5mC. By the use of ammonium bicarbonate (NH4HCO3) as an additive to the mobile phase, we improved the online-coupled MS/MS detection of 5mC, 5hmC, and 5-formylcytosine (5fC) by 1.8-14.3 times. The recovery of the method is approximately 100% for 5hmC, and 70-90% for 5mC. The relative standard deviation (RSD) of the interday precision is about 2.9-10.6%, and that of the intraday precision is about 1.4-7.7%. By the analysis of 13 volunteers using the developed method, we for the first time demonstrate the presence of 5hmC in human urine. Unexpectedly, we observed that the level of 5hmC (22.6 ± 13.7 nmol/L) is comparable to that of its precursor 5mC (52.4 ± 50.2 nmol/L) in human urine. Since the abundance of 5hmC (as a rare DNA base) is 1 or 2 orders of magnitude lower than 5mC in genomic DNA, our finding probably implicates a much higher turnover of 5hmC than 5mC in mammalian genomic DNA and underscores the importance of DNA demethylation in daily life.

Gadd45a promotes DNA demethylation through TDG

Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway.

Quantum Dots Enhanced Ultrasensitive Detection of DNA Adducts

Here we demonstrate that quantum dots (QD) can greatly improve the ultrasensitive capillary electrophoresis-laser induced fluorescence immunoassay of trace anti-benzo(a)pyrene diol epoxide (BPDE)-DNA adducts from sensitivity to separation. We for the first time show that the target QD-antibody-DNA complex is not only effectively separated but also effectively focused by capillary electrophoresis. With the online laser-induced fluorescence detection coupled, the low limits of detection of 6.6 x 10(-21) mol in mass and 120 fM in concentration are achieved for BPDE-DNA adducts. The achieved ultrasensitivity allows for human exposure biomonitoring and shows promising applications of QD in various DNA analyses, including DNA damage.

Potent DNA damage by polyhalogenated quinones and H2O2 via a metal-independent and Intercalation-enhanced oxidation mechanism

Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important ·OH can be produced by polyhalogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether this unusual metal-independent ·OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and H2O2 can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to ·OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and H2O2 can induce potent DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicity.

Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine

The gene- or fragment-specific detection of newly recognized deoxyribonucleic acid (DNA) base 5-hydroxymethylcytosine (5hmC) will provide insights into its critical functions in development and diseases, and is also important for screening 5hmC-rich genes as an indicator of epigenetic states, pathogenic processes and pharmacological responses. Current analytical technologies for gene-specific detection of 5hmC are heavily dependent on glucosylated 5hmC-resistant restriction endonuclease cleavage. Here, we find that boronic acid (BA) can inhibit the amplification activity of Taq DNA polymerase for replicating glucosylated 5hmC bases in template DNA by interacting with their glucose moiety. On the basis of this finding, we propose for the first time a BA-mediated polymerase chain reaction (PCR) assay for rapid and sensitive detection of gene- or fragment-specific 5hmC without restriction-assay-like sequence limitations. To optimize the BA-mediated PCR assay, we further tested BA derivatives and show that one BA derivative, 2-(2′-chlorobenzyloxy) phenylboronic acid, displays the highest inhibitory efficiency. Using the optimized assay, we demonstrate the enrichment of 5hmC in an intron region of Pax5 gene (a member of the paired box family of transcription factors) in mouse embryonic stem cells. Our work potentially opens a new way for the screening and identification of 5hmC-rich genes and for high throughput analysis of 5hmC in mammalian cells.

Nickel (II) inhibits Tet-mediated 5-methylcytosine oxidation by high affinity displacement of the cofactor iron (II)

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Capillary monolithic bioreactor of immobilized snake venom phosphodiesterase for mass spectrometry based oligodeoxynucleotide sequencing.

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.