Genome-Wide Comprehensive Characterization of the Role of Pinin in Transcription Regulation of Skeletal Myogenesis

The transcriptome is the complete set of RNA molecules in a cell, expression of which is finely and dynamically regulated to meet the cellular needs associated with a particular development or physiological state. While extensive efforts have been devoted to characterizing the transcription and post-transcriptional mechanisms that demarcate the transcriptome, global and genome-wide analysis remains one of the less well explored step in terms of its regulation and functional consequences. The recent advent of whole-transcriptome deep-sequencing technologies (e.g., RNA-seq), along with their capacity to simultaneously assay the entire transcriptome at the nucleotides precision, has promoted RNA transcription discoveries in a global and quantitative manner. The overall goal of this proposal is to decipher the way by which pinin (pnn), a nuclear speckle-associated SR-like protein involved in multifaceted RNA regulation, exert its effect on transcriptome regulation during skeletal myogenesis. Despite its seemingly ubiquitous expression and function indispensability (Leu and Ouyang, 2006; Leu et al., 2012), it presently remains unsolved whether or how pnn regulates its target genes in a context-dependent manner. In this proposal, we are attempting to establish an integrated, functional genomic-based system that will aid us in exploring two of the major questions regarding pnn: (1) the mechanism of pnn-mediated gene expression regulation, and (2) patho-physiological significance of such transcription role in skeletal myogenesis. To characterize the role that pnn plays in transcriptional regulation, it is first necessary to identify genes that are directly regulated by pnn. We aim to systematically pinpoint pnn targets in the differentiating mouse myoblasts by first, RNA immunoprecipitation (RIP) combined with RNA Seq (RIP-Seq) to capture at the transcriptome-wide level RNAs bound by pnn and second, the recently developed ChIP-Seq technique to determine the localization of transcriptional regulator binding within marnmalian genomes (Aim 1). Moreover detailed molecular mechanism underlying pnn-targeted gene expression will also be assessed by delineating promoter-associated histone modification status (Aim 2). Finally to faithfully comprehend the biological and functional significance of pnn and its associated transcription factors and targeted genes in skeletal myogenesis, we will employ pnn conditional knockout (ko) mouse model to demonstrate the outcomes derived from our in vitro culture studies (Aim 3). The results from conditional mouse model with pnn specifically deficient in skeletal muscle will not only validate the observation obtained from high throughput analysis, but also will fill the gap of data interpretation between cell-based study and in vivo animal model. By answering these questions with our integrated approaches, we can get a comprehensive picture of the biological significance of pnn and the transcription regulation via pnn in general.

Project ID:PC10601-0106
External Project ID:MOST104-2320-B182-011-MY3