Molecular mechanisms of the transcriptional regulation by PGC-1α/β in skeletal muscle

Skeletal muscle (SKM) is an energetic organ with a high degree of plasticity. Different environmental stimuli as exercise or cold, but also physical inactivity, lead to complex molecular regulations that result in metabolic adaptations of the SKM and the whole body. Key factors in SKM plasticity and whole body energy homeostasis are the peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 (PGC-1) family including three members, PGC-1α, PGC-1β and PGC-related coactivator (PRC). The PGC-1s are coactivators and hence use transcription factor binding partners (TFBP) in order to regulate their target genes. The complexity of transcriptional control might even be increased by epigenetic alterations, mainly DNA methylation.
The aim of my thesis was to study the regulation of global molecular mechanisms by SKM PGC-1α and PGC-1β leading to muscle plasticity in various environmental contexts. We combined diverse experimental, computational and multi-omics approaches such as chromatin immunoprecipitation sequencing (ChIPseq), RNA sequencing (RNAseq), reduced representation bisulfite sequencing (RRBS) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated proteins) genome editing technology in skeletal muscle systems in vitro and in vivo and investigated the effect of external stimuli as cold or exercise in different PGC-1α/β genotypes.
Our data show that various interventions like acute and chronic exercise have different methylation profiles or combined with cold-induced muscle shivering, individual transcript profiles in wild type (WT) mice. A time-dependent correlation of DNA methylation with gene expression was observed, however dissimilar in acute and chronic exercise. Furthermore, we dissected potential memory marks on the DNA by methylation following chronic training in mice. In addition, we could show for the first time a role of PGC-1α, not only in exercise performance but as well in altered transcriptome and methylome profiles subsequent to exercise and changed transcription profile to cold stimulation, by using muscle-specific PGC-1α knockout (MKO) mice. Thus, PGC-1α is a major contributor in global metabolic control by the regulation of a transcriptional network through multiple TF interactions and its involvement in epigenetic alterations. To further investigate the PGC-1α network, the Ppargc1a locus multiplex epitope tag knock-in mouse, which we generated by the CRISPR/Cas technology, will serve as a platform for future studies. This genetic mouse model allows now detailed evaluation of PGC-1α isoforms as well as the identification of new TFBPs under diverse contexts and in different tissues, due to non-tissue-specific epitope tags at the proximal PGC-1α promoter. However, in C2C12 myotubes we could show that PGC-1α regulates its target genes either by direct TF binding or indirectly. Even more, the genomic context of guanine-cytosine (GC) and cytosine-phosphate-guanine (CpG) amount affects PGC-1α recruitment and allows the estrogen-related receptor α (ERRα), a known TFBP of PGC-1α in the regulation of mitochondrial biogenesis, to regulate PGC-1α target genes without coactivation by PGC-1α but by interaction with the TF specificity protein 1 (SP1). Contrarily, we observed that PGC-1β acts mostly indirect on its target genes and only to a very small extent direct on the DNA by TF binding.
Taken together, our data provide new knowledge of the functional role of PGC-1α and PGC-1β in SKM metabolism. The involvement of transcriptional regulation and epigenetic control under basal, acute and chronic exercise conditions as well as in cold-induced muscle shivering, adds a next piece of puzzle to the complex network regulated by these coactivators. Our findings help to understand the mechanism of SKM plasticity and open new signaling pathways and targets, which will, complemented with further studies, support the development of novel therapeutic strategies to cure myopathies and fight against metabolic disorders and other pathophysiological conditions.