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
Project description:Global patterns of DNA methylation, mediated by the DNA methyltransferases (DNMTs), are disrupted in all cancers by mechanisms that remain largely unknown, hampering their development as therapeutic targets. Combinatorial acute depltion of all DNMTs in a pluripotent human tumor cell line, followed by epigenome and transcriptome analysis, revealed DNMT functions in unprecedented detail. DNMT3B occupancy regulates methylation during differentiation, while an unexpected interplay was discovered in which DNMT1 and DNMT3B antithetically regulate methylation and hydroxymethylation in gene bodies, a finding confirmed in other cell types. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCpG versus CpG site methylation. Taken together, these data reveal new functional targets of each DNMT suggesting that isoform selective inhibition would be therapeutically advantageous. NCCIT cells were transfected with siRNAs against DNMT3B and a no-target control. Genomic DNA was extracted, and subsequently applied to affinity MBD-bound magnetic beads (Ribomed) to enrich methylated DNA sequences.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
