Building a Gene Regulatory Network in Adult Mouse Skeletal Muscle Following Nerve Injury: Transcriptome Characterization and New Model for Functional cis-Regulatory Analysis In Vivo

Abstract

The essential functional linkages of gene regulatory networks (GRNs) consist of the interactions between cis-regulatory DNA sequences and trans-acting regulatory factors. These genomically encoded regulatory interactions govern the differential gene expression programs which direct specific biological processes during development and adulthood. Detailed analysis of GRNs during development has yielded important insights regarding the structural and functional dynamics of cis-regulatory modules (CRMs) and cis-regulatory elements (CREs). Indeed, the comprehensive GRNs that have been characterized for various developmental processes provide a model for both the methodological approach and the intellectual understanding required to explore cis-regulatory architecture in other biological contexts. The present study focuses on the physiological context, investigating the GRNs that govern the molecular response to nerve injury in adult mouse skeletal muscle. Until now, high-quality GRN investigations in this context have been hampered by the absence of two fundamental components: a comprehensive catalog of genes differentially expressed after nerve injury, and an effective in vivo gene transfer technique to functionally test putative cis-regulatory modules. Using RNAseq, we have compiled a comprehensive list of all differentially expressed genes at 6.0, 12.0, 24.0, and 168.0 hours following nerve injury. This data has validated previously known differentially expressed genes, as well as identified novel candidates for cis-regulatory analysis. The in vivo gene transfer technique I have adapted and advanced targets an easily accessible muscle group for minimally invasive injection and electroporation of DNA; with it, I demonstrate highly efficient, reproducible, and stable gene transfer in mouse skeletal muscle. In addition, I have optimized the gene transfer technique not only for plasmid DNA reporter vectors, but also for BAC DNA reporter vectors, thus enabling cis-regulatory modules to be tested in a broad chromosomal environment. Finally, I have validated the capacity of this gene transfer method to functionally test CRMs, by identifying a nerve injury-associated CRM of the skeletal muscle-specific myogenin gene. The enhanced resolution provided by this technique allowed for qualitative and quantitative detection of increased reporter signal from a mutated version the nerve injury-associated CRM at ten days following denervation, when compared to the wild-type CRM, implicating it as the cis-acting regulatory sequence responsible for mediating the down-regulation of myogenin during late phase neurogenic skeletal muscle atrophy. This work lays the foundation from which a high-quality adult skeletal muscle GRN can be constructed for nerve injury and other muscle-associated disease states.