Protonation-Suppression-Free LC-MS/MS Analysis for Profiling of DNA Cytosine Modifications in Adult Mice.

DNA cytosine modifications are important epigenetic marks. To elucidate their roles by a large scale of comparative studies, it is important to quantify the abundance of DNA cytosine modifications accurately. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a golden option. The performance of LC-MS/MS is heavily dependent on the ionization or protonation of target analytes. Initially, we found that two factors, DNA hydrolysate buffer and residual coeluted nucleosides, might greatly suppress the protonation of 5-(hydroxymethyl)-2'-deoxycytidine (5hmdC). Surprisingly, ammonium bicarbonate can eliminate the suppression caused by both factors. Mechanistically, ammonium bicarbonate increases the protonation capacity in the gas phase and facilitates proton transfer to the target nucleosides. Benefiting from these findings, we developed a suppression-free, sensitive, and robust ultrahigh-performance LC-MS/MS assay for massive detection of three DNA cytosine modifications, including 5-methyl-2'-deoxycytidine (5mdC), 5hmdC, and 5-formyl-2'-deoxycytidine (5fdC). In 30 consecutive analyses, the relative standard deviation (RSD) of the 5hmdC and 5fdC peak areas is 2.0% and 3.2%, respectively. In this case, no stable isotope-labeled standard is required for internal calibration. We further performed a comprehensive profiling of DNA cytosine modifications in 26 tissues of age-different C57BL/6N mice. Interestingly, we found that only liver 5hmdC abundance increases with the increasing age of adult mice, suggesting that liver 5hmdC might be a potential indicator of age in adulthood.

Global DNA 5-Hydroxymethylcytosine and 5-Formylcytosine Contents Are Decreased in the Early Stage of Hepatocellular Carcinoma.

Methylation of the fifth position of cytosine (5mC) is an important epigenetic modification of DNA. It has been shown that the oxidized derivatives of 5mC, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), are in dynamic existence and have distinct regulatory functions. In the current study, we investigated whether there are changes in the contents of all three 5mC-oxidized derivatives in the hepatocellular carcinoma (HCC) genome and further explored the underlying mechanisms. We showed that both global genomic 5hmC and 5fC contents were decreased significantly in the very early stage (stage 0, Barcelona Clinic Liver Cancer [BCLC] staging) of HCC compared with those of paratumor tissues. Noteworthily, 5fC content continued to decrease in the late stage (BCLC staging from 0 to A) of HCC. The 5caC content in HCC tissues was below the detection threshold. Hepatitis B virus (HBV) infection was associated with 5mC, 5hmC, or 5fC decrease in HCC; and measurements in cell lines integrated with or without HBV DNA showed consistent results. On the other hand, both the expression level of ten-eleven translocation enzyme 2 (TET2) and α-ketoglutarate content were decreased significantly in HCC. The significantly positive correlations among the expression levels of DNA methylation-related enzymes in paratumor tissues were generally attenuated or even disappeared in HCC tumor tissues. The decreases of both 5hmC and 5fC contents in genomic DNA were associated with poor prognosis of HCC patients. Conclusion: Global 5hmC and 5fC contents were decreased significantly in the very early stage of HCC; the decrease of 5hmC and 5fC was mainly due to the decrease of 5mC and associated with HBV infection, decreased TET enzyme activity, and uncoordinated expression of DNA methylation-related enzymes.

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.

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation.

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.

Structural insights into molecular mechanism for N6-adenosine methylation by MT-A70 family methyltransferase METTL4.

METTL4 belongs to a subclade of MT-A70 family members of methyltransferase (MTase) proteins shown to mediate N6-adenosine methylation for both RNA and DNA in diverse eukaryotes. Here, we report that Arabidopsis METTL4 functions as U2 snRNA MTase for N6-2'-O-dimethyladenosine (m6Am) in vivo that regulates flowering time, and specifically catalyzes N6-methylation of 2'-O-methyladenosine (Am) within a single-stranded RNA in vitro. The apo structures of full-length Arabidopsis METTL4 bound to S-adenosyl-L-methionine (SAM) and the complex structure with an Am-containing RNA substrate, combined with mutagenesis and in vitro enzymatic assays, uncover a preformed L-shaped, positively-charged cavity surrounded by four loops for substrate binding and a catalytic center composed of conserved residues for specific Am nucleotide recognition and N6-methylation activity. Structural comparison of METTL4 with the mRNA m6A enzyme METTL3/METTL14 heterodimer and modeling analysis suggest a catalytic mechanism for N6-adenosine methylation by METTL4, which may be shared among MT-A70 family members.

Nickel(ii) inhibits the oxidation of DNA 5-methylcytosine in mammalian somatic cells and embryonic stem cells.

Nickel is found widely in the environment. It is an essential microelement but also toxic. However, nickel displays only weak genotoxicity and mutagenicity. Exploration of the epigenetic toxicity of nickel is extremely interesting. Iron(ii)- and 2-oxoglutarate-dependent Tet dioxygenases are a class of epigenetic enzymes that catalyze the oxidation of DNA 5-methylcytosine (5mC). Thus, they are critical for DNA demethylation and, importantly, are involved with nuclear reprogramming, embryonic development, and regulation of gene expression. Here, we demonstrated that nickel(ii) dramatically inhibits Tet proteins-mediated oxidation of DNA 5mC in cells ranging from somatic cell lines to embryonic stem cells, as manifested by the consistent observation of a significant decrease in 5-hydroxymethylcytosine, a critical intermediate resulting from the oxidation of 5mC. The inhibitory effects of nickel(ii) were concentration- and time-dependent. Using HEK293T cells overexpressing Tet proteins and ascorbic acid-stimulated Tet-proficient ES cells, we observed that nickel(ii) significantly reduced DNA demethylation at the global level. Interestingly, we also showed that nickel(ii) might affect the naïve or ground state of pluripotent embryonic stem cells. Here we show, for the first time, that nickel(ii) represses the oxidation of DNA 5mC and potentially alters the Tet proteins-regulated DNA methylation landscape in human cells. These findings provide new insights into the epigenetic toxicology of nickel.

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.

Facile synthesis of magnetic molecularly imprinted polymers and its application in magnetic solid phase extraction for fluoroquinolones in milk samples.

In this work, we proposed a simple co-mixing method to fabricate magnetic molecularly imprinted polymers (magnetic MIPs). MIPs were commercial products while magnetic nanoparticles (MNPs) were prepared by chemical oxidation and solvothermal methods. When MNPs and MIPs (with mass ratio 1:1) were co-mixed and vortexed evenly in methanol, they could assemble into magnetic composites spontaneously and thus be magnetically separable. To testify the feasibility of the magnetic composites in sample preparation, the resultant magnetic MIPs were applied as sorbents for magnetic solid-phase extraction (MSPE) of fluoroquinolones (FQs) in milk samples. Under optimized conditions, a rapid, convenient, and efficient method for the determination of three FQs in milk samples was established by magnetic MIPs based MSPE coupling with high performance liquid chromatography with ultraviolet detector (HPLC-UV). The limits of detection (LODs) for three FQs were found to be 1.8-3.2ng/g. The intra- and inter-day relative standard deviations (RSDs) were less than 9.5% and 12.5%, respectively. The recoveries of FQs for two spiked milk samples were in the range from 94.0% to 124.4% with the RSDs less than 11.6%.

Quick, easy, cheap, effective, rugged and safe method with magnetic graphitized carbon black and primary secondary amine as adsorbent and its application in pesticide residue analysis.

By using magnetic graphitized carbon black and primary secondary amine (GCB/PSA/MNPs) as adsorbent, a modified quick, easy, cheap, effective, rugged and safe (QuEChERS) method was proposed for pesticide residue analysis in vegetables. The magnetic adsorbent was fabricated via simple co-mixing method based on an "aggregate warp" mechanism. To achieve the optimum conditions of modified QuEChERS toward target analytes, several parameters, including the composition of analyte protectants and the amount of the adsorbents were investigated. Under the optimized conditions, a simple, rapid and effective method for the determination of 10 pesticide residues in vegetables was established by coupling modified QuEChERS to gas chromatography/mass spectrometry analysis. The detection limits of the proposed method for 10 pesticides ranged from 0.39 to 8.6ng/g. Good linearity (R value≥0.990) was achieved at concentration levels of 10-200ng/g, and acceptable method reproducibility was found as intra- and inter-day precisions, yielding the relative standard deviations less than 10.7% and 13.4%, respectively. The recoveries were in the range of 69.9-125.0% at different concentrations for real samples. Compared with the reported methods for the determination of a large number of samples, the proposed method has the advantage of less time-consuming in clean-up procedure.