Mutagenic and Cytotoxic Properties of Oxidation Products of 5-Methylcytosine Revealed by Next-Generation Sequencing

<div><p>5-methylcytosine (5-mC) can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and finally to 5-carboxylcytosine (5-caC), which is thought to function in active DNA cytosine demethylation in mammals. Although the roles of 5-mC in epigenetic regulation of gene expression are well established, the effects of 5-hmC, 5-foC and 5-caC on DNA replication remain unclear. Here we report a systematic study on how these cytosine derivatives (5-hmC, 5-foC and 5-caC) perturb the efficiency and accuracy of DNA replication using shuttle vector technology in conjugation with next-g sequencing. Our results demonstrated that, in <i>Escherichia coli</i> cells, all the cytosine derivatives could induce CT transition mutation at frequencies of 0.17%–1.12%, though no effect on replication efficiency was observed. These findings provide an important new insight on the potential mutagenic properties of cytosine derivatives occurring as the intermediates of DNA demethylation.</p></div

Diversity-Oriented Synthesis of Coumarin-Linked Benzimidazoles via a One-Pot, Three-Step, Intramolecular Knoevenagel Cyclization

Diversity-oriented synthesis of coumarin-linked benzimidazoles from <i>N</i>-(2-aminophenyl)-2-cyanoacetamide was achieved via a one-pot, three-step sequential reaction in excellent yields. In situ intramolecular cyclization of the cyanoacetamide afforded benzimidazoles which subsequently underwent a Knoevenagel condensation of the 2-cyanomethylbenzimidazoles with salicylaldehydes promoted by triethylamine to reach the target compounds. An important intermediate, 2-(2-imino-2<i>H</i>-chromen-3-yl)-1<i>H</i>-benzimidazole was characterized by X-ray analysis and further hydrolyzed to 2-(coumarin-3-yl)­benzimidazole in acidic condition. Among the synthesized compounds, some were found to be promising inhibitors of porcine kidney d-amino acid oxidase (<i>pk</i>DAO)

Bypass efficiencies and mutation frequencies of cytosine derivatives.

<p>(A) Bypass efficiencies of 5-hmC, 5-foC and 5-caC. (B–E) Mutation frequencies of control (B), 5-hmC (C), 5-foC (D) and 5-caC (E). The data represent the means and standard deviations of results from three independent experiments.</p

Effects of circadian clock genes and health-related behavior on metabolic syndrome in a Taiwanese population: Evidence from association and interaction analysis

<div><p>Increased risk of developing metabolic syndrome (MetS) has been associated with the circadian clock genes. In this study, we assessed whether 29 circadian clock-related genes (including <i>ADCYAP1</i>, <i>ARNTL</i>, <i>ARNTL2</i>, <i>BHLHE40</i>, <i>CLOCK</i>, <i>CRY1</i>, <i>CRY2</i>, <i>CSNK1D</i>, <i>CSNK1E</i>, <i>GSK3B</i>, <i>HCRTR2</i>, <i>KLF10</i>, <i>NFIL3</i>, <i>NPAS2</i>, <i>NR1D1</i>, <i>NR1D2</i>, <i>PER1</i>, <i>PER2</i>, <i>PER3</i>, <i>REV1</i>, <i>RORA</i>, <i>RORB</i>, <i>RORC</i>, <i>SENP3</i>, <i>SERPINE1</i>, <i>TIMELESS</i>, <i>TIPIN</i>, <i>VIP</i>, and <i>VIPR2</i>) are associated with MetS and its individual components independently and/or through complex interactions in a Taiwanese population. We also analyzed the interactions between environmental factors and these genes in influencing MetS and its individual components. A total of 3,000 Taiwanese subjects from the Taiwan Biobank were assessed in this study. Metabolic traits such as waist circumference, triglyceride, high-density lipoprotein cholesterol, systolic and diastolic blood pressure, and fasting glucose were measured. Our data showed a nominal association of MetS with several single nucleotide polymorphisms (SNPs) in five key circadian clock genes including <i>ARNTL</i>, <i>GSK3B</i>, <i>PER3</i>, <i>RORA</i>, and <i>RORB</i>; but none of these SNPs persisted significantly after performing Bonferroni correction. Moreover, we identified the effect of <i>GSK3B</i> rs2199503 on high fasting glucose (P = 0.0002). Additionally, we found interactions among the <i>ARNTL</i> rs10832020, <i>GSK3B</i> rs2199503, <i>PER3</i> rs10746473, <i>RORA</i> rs8034880, and <i>RORB</i> rs972902 SNPs influenced MetS (P < 0.001 ~ P = 0.002). Finally, we investigated the influence of interactions between <i>ARNTL</i> rs10832020, <i>GSK3B</i> rs2199503, <i>PER3</i> rs10746473, and <i>RORB</i> rs972902 with environmental factors such as alcohol consumption, smoking status, and physical activity on MetS and its individual components (P < 0.001 ~ P = 0.002). Our study indicates that circadian clock genes such as <i>ARNTL</i>, <i>GSK3B</i>, <i>PER3</i>, <i>RORA</i>, and <i>RORB</i> genes may contribute to the risk of MetS independently as well as through gene-gene and gene-environment interactions.</p></div

The sequences of the 27mer cytosine derivative-containing and the control ODNs used for replication studies.

<p>‘X’ designates modified cytosine and the barcode is underlined in each 27mer sequence.</p

The structures of the cytosine derivatives.

<p>5-mC modification is catalyzed by DNA methyltransferase (DNMT). 5-mC can be demethylated through the oxidation of 5-mC by TET proteins to produce 5-hmC, 5-fC and 5-caC. TDG, thymine-DNA glycosylase; BER, base-excision repair.</p

Demographic and clinical characteristics of study subjects.

<p>Demographic and clinical characteristics of study subjects.</p

Odds ratio analysis with odds ratios after adjustment for covariates (including age and gender) between the MetS and 16 SNPs in five selective circadian clock genes, which have nominal evidence of association (P < 0.01).

<p>Odds ratio analysis with odds ratios after adjustment for covariates (including age and gender) between the MetS and 16 SNPs in five selective circadian clock genes, which have nominal evidence of association (P < 0.01).</p

Gene-environment interaction models identified by the GMDR method with adjustment for age and gender.

<p>Gene-environment interaction models identified by the GMDR method with adjustment for age and gender.</p

Two-way gene-gene interaction models by using the GMDR method with adjustment for age and gender.

<p>Two-way gene-gene interaction models by using the GMDR method with adjustment for age and gender.</p