Large-scale identification of transcriptional networks during myogenesis
Stem cells play a central role in development and maintenance of tissues and organs in the body of animals and humans. During growth or regeneration, skeletal muscle cells are unable to divide but replenish from a population of progenitor stem cells, which have the unique ability to divide, to produce copies of themselves as well as differentiating muscle cells. F. Relaix has identified and characterized a novel major progenitor stem cell population which gives rise to nearly all skeletal muscle cells, including the myogenic stem cell population of the adult, and identified key transcription regulators (Pax3 and Pax7) implicated in survival, specification and proliferation of these cells.
The aim of the project is to identify the molecular transcriptional mechanisms of myogenic progression in vivo. The central strategy is to develop a tight collaboration between the group of F. Relaix, where all the biological data will be generated and validated, and the team of O. Poch , where all the computing analysis will be performed. The project will primarily utilize the mouse, as this is the only mammalian system tractable for comprehensive molecular genetic studies.
As a first step, the laboratory of F. Relaix is currently generating new mouse genetic tools (transgenic mice carrying fluorescent reporter genes targeted in genes marking key step of myogenic progession) in order to gain access to pure myogenic populations in vivo using cell sorting. Using this strategy we will be able to isolate muscle progenitor cells, myoblasts and fibers at different time-points (throughout development, in postnatal and regenerating muscles) and perform transcriptomal analyses in collaboration with O. Poch’s team. Large-scale quantitative RT-PCR will be undertaken to validate the microarrays data. This high-throughput transcriptomal analysis will provide us with the complete set of genes involved in the myogenic lineage.
In parallel, the team of O. Poch will characterize of the total set of mouse proteins involved directly or indirectly in the transcriptional processes. This will require an in depth sequence, structural, evolutionary (SSE) and functional analysis of the mouse proteome with the major objective of defining and delineating any conserved domains or regions that might be associated to known transcriptional modules. This work will be performed in collaboration with M. Andrade’s team (Ottawa, Canada) in the context of the International Regulome Consortium (http://www.internationalregulomeconsortium.ca/). In the framework of the proposed Decrypthon project, the SSE analysis of the entire human/mouse proteome (~60 000 proteins including splice variants and the human or mouse specific proteins) will involve a pipeline of processes starting with homology identification, multiple sequence alignment, structural and functional subfamily classification, orthology/paralogy analysis and phylogenetic reconstruction. We will take advantage of the previous developments performed on the Decrypthon grid, notably those concerning the MACSIMS (Multiple Alignment of Complete Sequence Information Management System) functional annotation and new protocols will be developed including PSI-Blast searches to detect distantly related proteins, recent multiple alignment algorithms implementation and phylogenetic tree algorithms. Protocols ensuring automated updating and storage in a relational database, hosted by the Decrypthon, will be developed.
The results will be combined with the data from the transcriptomal analysis performed in vivo. This complementary approach is expected to help us to identify and characterise the transcriptional networks involved in muscle development, specification, regeneration and myogenic progression. In vivo functional validation will be done using mouse molecular genetics and expertise in muscle biology in the laboratory of F. Relaix.