Project description:We performed a CRISPR-based functional genetic screen targeting TEAD4 binding motif located within the YAP-bound enhancers. The screen identified seven functional enhancers whose targeting resulted in a marked negative effect on the proliferation of MCF10A-YAP-5SA cells (overexpressed constrictively activated YAP) but no significant effect on MCF10A (parental control) cells. We then carried out RNA-seq analysis on MCF10A-YAP-5SA cells transduced with sgRNA vectors targeting these seven enhancers (enhancers A, B, C, D, E, F, G) as well as the non-targeting sgRNA as control (NT).
Project description:To understand the direct target genes YAP transcription co-activator invovled with in gonome-wide levels, next generation sequencing was conducted in Flag-YAP-5SA overexpressing MCF10A cells using Flag (sigma) antibody.
Project description:Aim of the study was to elucidate YAP-regulated trophoblast-specific genes by analyzing differentially-expressed gene signatures between first trimester human cytotrophoblasts (vCTBs) transfected with a YAP-5SA mutant or a control plasmid.
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:To investigate whether YAP upregulates MYC via miRNA repression, we sequenced miRNAs from gastric epithelial cells (HFE-145) either expressed with vector or YAP-5SA.
Project description:Asthma is a chronic inflammatory airway disease characterized by airway inflammation and remodeling. The role of 15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-oxoETE), a 15-HETE metabolite catalyzed by 15-prostaglandin dehydrogenase (15-PGDH), has been relatively unexplored in asthma. In this study, we used RNA-seq to explore the effect of 15-KETE on the transcriptome of airway epithelial cells, aiming to identify its potential downstream targets and mechanisms of action.