Project description:Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4
Project description:Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4 Cells were treated with 10 mM butyrate for 24 hr, The cells were scraped from the flask and homogenized with ice-cold Dounce homogenizer to release the nuclei. The collected nuclei were resuspended in digestion buffer and enzymatic shearing was performed. ChIP with anti H3, H4 and acetyl-H3 and acetyl-H4.
Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.
