Project description:DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions, that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule and thus, single cell level that can be used to monitor disease progression and response to therapy. Nucleosome Occupancy and Methylome-Sequencing (NOMe-Seq) on IMR90 cell line and 2 GBM cell lines
Project description:Nucleosome positioning in a 2.3 Mb region of human chromosome 12 (chr12: 6,140,000-8,460,000) containing GAPDH and NANOG loci in human IMR90 fibroblasts (hFibs) and fibroblast-derived human induced pluripotent stem cell (hiPSCs).
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes