Biochemical and Biophysical Research Communications
Regular ArticleA Sensitive New Method for Rapid Detection of Abnormal Methylation Patterns in Global DNA and within CpG Islands
Abstract
To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5′ guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [3H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [3H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The “cytosine-extension assay” has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.
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Molecular Techniques for DNA Methylation Studies
2017, Molecular Diagnostics: Third EditionDNA methylation is the best-studied epigenetic modification and altered DNA methylation patterns have been identified in cancer and many other complex diseases. The first DNA methylation-based biomarkers for early diagnosis of cancer have been approved by regulatory agencies, which illustrate the potential and advantages of DNA methylation analysis. A number of technologies have been devised to study DNA methylation genome-wide or at specific loci using a limited number of principles for differentiating the methylation state (methylation-sensitive/dependent restriction enzymes, antibody or methyl-binding protein, or bisulfite conversion). The choice of the best-suited assay depends on the biological question to be investigated, the quantity and quality of biological material available, and access to specific instrumentation. For the discovery of altered methylation patterns, second-generation sequencing has largely replaced microarrays as a read-out platform whereas for locus-specific DNA methylation analysis, a number of robust and quantitative technologies are available that deliver accurate DNA methylation profiles at single-nucleotide resolution. In this chapter, currently used methods for genome-wide as well as locus-specific analysis of 5-methylcytosine are reviewed in detail and advantages and limitations of each approach are discussed.
Effects of oral exposure to bisphenol A on gene expression and global genomic DNA methylation in the prostate, female mammary gland, and uterus of NCTR Sprague-Dawley rats
2015, Food and Chemical ToxicologyBisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4–5 orders of magnitude lower than that of estradiol. We reported previously that “high BPA” [100,000 and 300,000 µg/kg body weight (bw)/day], but not “low BPA” (2.5–2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The “high BPA” effects partially overlapped those of ethinyl estradiol (EE2, 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of “low BPA” to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the “high BPA” effects with those of EE2. The “low BPA” doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes.
Hypermethylated ERG as a cell-free fetal DNA biomarker for non-invasive prenatal testing of Down syndrome
2015, Clinica Chimica ActaPrevious reports have shown that the ERG gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. In this study, we explore the feasibility of hypermethylated ERG as a cell-free fetal (cff) DNA biomarker for non-invasive prenatal testing (NIPT) of Down syndrome.
We randomly selected 90 healthy pregnant women, including 30 cases at each trimester of pregnancy. In addition, 15 pregnant women were recruited as the case group whose fetuses had been confirmed to have trisomy 21 by amniotic fluid analysis at 18th to 26th week gestation. Using HpaII, MspІ to digest cell-free maternal plasma DNA, we performed SYBR Green PCR to detect methylated sites of ERG sequences, and analyzed the concentrations of cff DNA in maternal plasma in different gestational trimesters and the case group.
The ERG median concentrations of the maternal plasma after Hpa II digestion (LG copies/ml) in first, second and third-trimesters were 5.38, 6.10, and 7.04, respectively (Kruskal–Wallis, P < 0.01); and that in the trisomy 21 case group was 6.85, which was higher than the second-trimester (Mann–Whitney, P < 0.01).
The study demonstrated that ERG gene is hypermethylated in cff DNA but hypomethylated in maternal DNA; and the median concentration of ERG gene in the trisomy 21 case group is higher than that in the gestational trimester matched normal group. ERG gene, as a fetal DNA biomarker, may be useful for NIPT of Down syndrome.
Gene expression and epigenetic profiles of mammary gland tissue: Insight into the differential predisposition of four rat strains to mammary gland cancer
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Tamoxifen is a non-steroidal anti-estrogenic drug widely used for the treatment and prevention of breast cancer in women; however, there is evidence that tamoxifen is hepatocarcinogenic in rats, but not in mice. Additionally, it has been reported that tamoxifen may cause non-alcoholic fatty liver disease (NAFLD) in humans and experimental animals. The goals of the present study were to (i) investigate the mechanisms of the resistance of mice to tamoxifen-induced hepatocarcinogenesis, and (ii) clarify effects of tamoxifen on NAFLD-associated liver injury. Feeding female WSB/EiJ mice a 420 p.p.m. tamoxifen-containing diet for 12 weeks resulted in an accumulation of tamoxifen-DNA adducts, (E)-α-(deoxyguanosin-N2-yl)-tamoxifen (dG-TAM) and (E)-α-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-DesMeTAM), in the livers. The levels of hepatic dG-TAM and dG-DesMeTAM DNA adducts in tamoxifen-treated mice were 578 and 340 adducts/108 nucleotides, respectively, while the extent of global DNA and repetitive elements methylation and histone modifications did not differ from the values in control mice. Additionally, there was no biochemical or histopathological evidence of NAFLD-associated liver injury in mice treated with tamoxifen. A transcriptomic analysis of differentially expressed genes demonstrated that tamoxifen caused predominantly down-regulation of hepatic lipid metabolism genes accompanied by a distinct over-expression of the lipocalin 13 (Lcn13) and peroxisome proliferator receptor gamma (Pparγ), which may prevent the development of NAFLD. The results of the present study demonstrate that the resistance of mice to tamoxifen-induced liver carcinogenesis may be associated with its ability to induce genotoxic alterations only without affecting the cellular epigenome and an inability of tamoxifen to induce the development of NAFLD.
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To whom correspondence should be addressed at Division of Biochemical Toxicology, NCTR, 3900 NCTR Road, Jefferson, AR 72079. Fax: (870) 543-6620. E-mail: [email protected].