MAGI: Methylation analysis using genome information

By incorporating annotation information into the analysis of next-generation sequencing DNA methylation data, we provide an improvement in performance over current testing procedures. Methylation analysis using genome information (MAGI) is applicable for both unreplicated and replicated data, and provides an effective analysis for studies with low sequencing depth. When compared with current tests, the annotation-informed tests provide an increase in statistical power and offer a significance-based interpretation of differential methylation.     

DNA repair modulates the vulnerability of the developing brain to alkylating agents

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag^?/?) or O⁶-methylguanine methyltransferase (Mgmt^?/?), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt^?/? neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag^?/? neurons were for the most part significantly less sensitive than wild type or Mgmt^?/? neurons to MAM and HN2. Aag^?/? neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt^?/? mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag^?/? or MGMT-overexpressing (Mgmt^Tg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt^Tg+ mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

Mechanism-based inhibition of lactoperoxidase by thiocarbamide goitrogens. Identification of turnover and inactivation pathways

Direct evidence is presented in support of mechanism-based (suicide) inactivation of lactoperoxidase by thiocarbamide thyroid inhibitors. The turnover of 1-methylbenzimidazolidine-2-thione was demonstrated by identifying the inhibitor-derived products 1-methylbenzimidazole and bisulfite ion that are formed concurrent to enzyme inactivation. The turnover of a hydroperoxide cosubstrate, 5-phenyl-4-pentenyl hydroperoxide, was quantitated from formation of the corresponding alcohol during enzyme inactivation. A specific inactivation pathway is suggested by the covalent binding of 1 mol of 14C- and 35S-labeled benzimidazolidine-2-thione and 1-methylbenzimidazolidine-2-thione per mole of inactivated lactoperoxidase. These results are explained by partitioning of inhibitor-derived S-oxygenated intermediates between turnover and inactivation pathways. The properties of the inactivation process are unique among thiono-sulfur compounds and suggest that benzimidazolinesulfenic acids are the reactive intermediates.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Identification of novel high-frequency DNA methylation changes in breast cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.     

Interacting quantitative trait loci control loss of peripheral tolerance and susceptibility to autoimmune ovarian dysgenesis after day 3 thymectomy in mice

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by CD4(+)CD25(+) T cells and the development of autoimmune ovarian dysgenesis (AOD) in A/J and (C57BL/6J x A/J)F(1) (B6AF(1)) hybrids but not in C57BL/6J mice. Quantitative trait loci (QTL) linkage analysis using a B6AF(1) x C57BL/6J backcross population verified Aod1 and Aod2 that were previously mapped as qualitative traits. Additionally, three new QTL intervals, Aod3, Aod4, and Aod5, on chromosomes 1, 2, and 7, respectively, influencing specific subphenotypes of AOD were identified. QTL linkage analysis using the A x B and B x A recombinant inbred lines verified Aod3 and confirmed linkage to H2. Aod5 colocalized with Mater, an ovarian-specific autoantigen recognized by anti-ovarian autoantibodies in the sera of D3Tx mice. Sequence analysis of Mater identified allelic, strain-specific splice variants between A/J and C57BL/6J mice making it an attractive candidate gene for Aod5. Interaction analysis revealed significant epistatic effects between Aod1-5 and Gasa2, a locus associated with susceptibility to D3Tx-induced autoimmune gastritis, as well as with H2. These results indicate that the QTL controlling D3Tx-induced autoimmune phenomenon are both organ specific and more generalized in their effects with respect to the genesis and activity of the immunoregulatory mechanisms maintaining peripheral tolerance.     

Understanding mechanisms of novel gene expression in polyploids

Polyploidy has long been recognized as a prominent force shaping the evolution of eukaryotes, especially flowering plants. New phenotypes often arise with polyploid formation and can contribute to the success of polyploids in nature or their selection for use in agriculture. Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through increased variation in dosage-regulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes. New research approaches are being used to study these mechanisms and the results should provide a more complete understanding of polyploidy.