AbstractsBiology & Animal Science

The troponin I fast (TnIfast) gene, a member of a differentially-expressed three-member TnI multigene family is expressed specifically in fast skeletal muscle fibers. The tissue and fiber type specificity of the TnIfast gene is driven by an Intronic Regulatory Element (IRE) in the first intron. The IRE is a 148 bp transcriptional enhancer, that contains four known or suspected cis regulatory elements; E-box, MEF2, CCAC, and CAGG elements. A previous study in this lab suggested that fast-fiber type specificity is driven by elements that reside in the 5'-most 30 bp of the IRE, a region that includes the E-box motif. My initial goal was to further localize the hypothetical fast fiber type specific element(s) within this region. The experimental approach was to make IRE partial deletions and mutations in reporter gene constructs in which IRE derivatives were cloned upstream of an enhancer-dependent TnI fast minimal promoter driving the reporter gene LacZ. The transcriptional activity of these constructs in fast and slow muscle fibers was evaluated by direct gene transfer into adult mouse skeletal muscle followed by histochemical analysis of LacZ reporter expression. My results showed that the E-box was not required for IRE fast-fiber specificity or high-level expression in adult skeletal muscles. Moreover, additional deletion constructs indicated, in contrast with the previous study, that the 5'-most 30 bp segment is not required for fast-fiber-specificity. I was able to show that a 49 bp IRE segment, not including the 5'-most 30 bp, but including MEF2 and CCAC elements, is sufficient to drive fast-fiber specific expression in adult mouse skeletal muscles. I discuss possible causes for the discordant results between the two studies, and the implications of my findings for the regulatory mechanisms of the IRE. Le gène troponin I rapide (TnIfast) est un member d'une famille de gènes multiple don't les members s'expriment d'une façons differente celon le type de fibre du muscle squelettique. La spécificité pour le type de tissue et le type de fibre du gène TnIfast est due a un "intronic regulatory element" (IRE) situé dans le premier intron. Le IRE est un enhancer de 148 bp qui contient quatre éléments-cis connus; E-box, MEF2, CCAC et le CAGG elements. Une étude précédente dans notre laboratoire a suggeré que la spécificité du gène troponin I rapide pour le type de fibre est due a des éléments résidant dans les 30 pair de bases situé dans l'éxtrimité gauche du IRE, une région qui contient le site "E-box". Mon but initial était de localise cet élément hypothétique dans ces 30 pairs de bases de l' IRE. Mon approche experimental était de préparer des versions du IRE mutées ou tronquées partiellement. Ces derives de IRE ont été couplés a un gène signal consistant en un promoter TnI fast minimal lié avec le gène LacZ. L'activité transcriptionnelle de ces construits été evalué par transfert de gène in vivo dans le muscle squelettique de souris…