SummarySkeletal muscle is an extremely plastic tissue that can undergo atrophy and hypertrophy, and even change its metabolism when stimulated by distinct challenges. The PGC-1a transcriptional coactivators have been shown to be important for the regulation of many of these adaptations. We have recently identified PGC-1a4 as a novel variant of the coactivator, specifically regulated by resistant-type exercise, that increases skeletal muscle size and strength. The aim of this project is to better understand the molecular mechanisms of action that confer PGC-1a4 its hypertrophic activity. The project was initiated six months ago, by completing the generation of some of the reagents that will be used in the next steps. At the moment, the PhD student in my group who is developing this project (Manziheh Izadi) is in Prof. Robert Roeder’s laboratory at The Rockefeller University, for a short period and in the context of a collaboration between our two laboratories to complete the next phase of the proposed project.
Scientific Progress Report
We have subcloned the PGC-1a4 cDNA into the appropriate vectors that will allow for protein expression and purification using bacterial systems and baculovirus-mediated delivery to insect cells. First expression and purification tests in bacteria were successful, so the GST-PGC-1a4 protein can be efficiently expressed and purified in bacteria. The PhD student developing the project is currently in Prof. Robert Roeder’s laboratory (The Rockefeller University), for a period of 5 months. Prof. Roeder is a world leader and reference in protein biochemistry, especially related to transcriptional regulation of gene expression. At the end of this internship, the student will return to my lab and transfer reagents and skills acquired during her visit. The next step of the project will be to use the different purified proteins to isolate and characterize PGC-1a4-associated protein-complexes from nuclear extracts.
We have been optimizing the technique to perform chromatin immunoprecipitation (ChIP) assays from mouse muscle. We have now a validated detailed protocol to perform this technique and are ready to use our Flag-PGC-1a4 transgenic mice to proceed with this part of the project. We will in parallel perform ChIP using mouse primary myotubes overexpressing the Flag-PGC-1a4 protein. By combining the information obtained for the biochemistry approach (identification of PGC-1a4 binding partners), with the genomics approach, we will begin to understand the gene networks under PGC-1a4 control.Jorge Ruas