Temporal profiling in muscle regeneration: Identification of novel transcriptional regulatory pathways.

摘要

Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression in a 6 time point cardiotoxin (CTX) degeneration/regeneration series in mouse muscle in vivo. Nucleated clusters with known downstream targets based on the expression pattern were further queried by promoter database mining and gel shift assay; Slug and calpain6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown as a downstream target by chromatin immunoprecipitation assay, promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice. We then continued the temporal expression profiling approach with expression profiling of 27 time points to more finely define temporally regulated gene clusters.; We further tested the identification of novel coordinately regulated genes by temporal clustering. A gene cluster temporally, coordinately regulated with myogenin was used as an example. Of the twenty genes/ESTs in this cluster, we found genes known to be upregulated during satellite cell activation and differentiation. Many other cluster members are likely to be novel genes expressed in satellite cell differentiation.; We then tested identification of potential downstream targets of Slug, a transcriptional repressor, by comparing expression profiles of muscle regeneration in Slug null mice and normal mice. Slow muscle genes were shown to be potentially negatively regulated by Slug.; We also sought to begin to define the differential molecular pathophysiology of severe and progressive Duchenne muscular dystrophy (DMD), vs. the mild and non-progresive homologous mouse model (mdx), by cross-species comparison of expression profiles. A number of transcripts whose levels differed between the mdx and human DMD were identified. These discrepancies provide candidates for salvage pathways that maintain skeletal muscle integrity in the absence of a functional dystrophin protein in mdx skeletal muscle.; Taken together, the data presented here provide the first genome-wide assessment of transcriptional activity in tissue regeneration and dystrophy in any disease system. Moreover, we prove the ability to use these data to construct transcriptional regulatory pathways and tissue salvage pathways.