MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N 12 ∕N 16 ) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers m CWG but not m CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies. 5-methylcytosine | methylomeM odified DNA bases appear in genomic DNAs in all domains of life, spanning the evolutionary distance from viruses to eukaryotic species. DNA base modifications vary in form and genomic location enriching the information content encoded by genomes. The biological role of base modifications varies, ranging from protection against restriction endonucleases in bacteria and bacteriophages to transcriptional regulation in mammals. In prokaryotes, DNA methyltransferases in restriction-modification systems modify the host genomic DNA, so that restriction endonucleases can target foreign DNA and protect the host cell from invaders (1). However, a few bacteriophages respond by incorporating modified bases into their genomes as a way to block restriction endonuclease cleavage (2). For example, in Xanthomonas oryzae phage XP12, all cytosines exist in the form of 5-methylcytosine (5mC) (3). Another example is the well-studied T4 phage, in which 5-hydroxymethylcytosine (5hmC) is incorporated into the DNA during replication and additional glucosyltransferases further modify all 5hmC to glucosylated-hydroxymethylcytosine (5ghmC). T4 genomic DNA containing 5ghmC is resistant to cleavage by most restriction endonucleases, with the exception of Type IV modification-dependent endonucleases (4, 5).Several different types of modification-dependent endonucleases are found in prokaryotes. For example, N6-adenosine methylation is recognized by a few known enzymes, e.g., DpnI (G m ATC). A group of sequence-specific cytosine methylationdependent restriction endonucleases including GlaI (G m CG m C), BisI (G m CNGC), etc., have been reported recently, which cleave within the recognition site in a Type IIP-like ...
IL-10 is a key pleiotropic cytokine that can both promote and curb Th2-dependent allergic responses. Herein we demonstrate a novel role for IL-10 in promoting mast cell expansion and the development of IgE-mediated food allergy. Oral ovalbumin challenge in sensitized BALB/c mice resulted in a robust intestinal mast cell response accompanied by allergic diarrhea, mast cell activation, and a predominance of Th2 cytokines, including enhanced IL-10 expression. In contrast, the development of intestinal anaphylaxis including diarrhea, mast cell activation, and Th2 cytokine production was significantly attenuated in IL-10−/− mice compared to WT controls. IL-10 also directly promoted the expansion, survival, and activation of mast cells, increased FcɛRI expression on mast cells, and enhanced the production of mast cell cytokines. IL-10−/− mast cells had reduced functional capacity, which could be restored by exogenous IL-10. Similarly, attenuated passive anaphylaxis in IL-10−/− mice could be restored by IL-10 administration. The adoptive transfer of WT mast cells restored allergic symptoms in IL-10−/− mice, suggesting that the attenuated phenotype observed in these animals is due to a deficiency in IL-10-responding mast cells. Lastly, transfer of WT CD4 T cells also restored allergic diarrhea and intestinal mast cell numbers in IL-10−/− mice, suggesting that the regulation of IL-10-mediated intestinal mast cell expansion is T cell-dependent. Our observations demonstrate a critical role for IL-10 in driving mucosal mast cell expansion and activation, suggesting that in its absence, mast cell function is impaired, leading to attenuated food allergy symptoms.
Herein we report a novel approach for fast, label-free probing of DNA-histone interactions in individual nucleosomes. We use solid-state nanopores to unravel individual DNA/histone complexes for the first time, and find that the unraveling time depends on the applied electrophoretic force, and our results are in line with previous studies that employ optical tweezers. Our approach for studying nucleosomal interactions can greatly accelerate our understanding of fundamental mechanisms by which transcription, replication, and repair processes in a cell are modulated through DNA-histone interactions, as well as in diagnosis of diseases with abnormal patterns of DNA and histone modifications.
We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system. (Mol Cancer Res 2008;6(8):1365 -74)
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