Troponin I (TnI), like many striated muscle contractile proteins, consists of multiple isoforms encoded by a multigene family whose members are differentially expressed in the different striated muscle cell types. Two TnI genes, TnIfast and TnIslow, are expressed in skeletal muscle the former in fast muscle fibers, the latter in slow fibers. The tissue- and fiber-type-specificities of the TnI fast and slow genes are driven by transcriptional enhancer elements: a Slow Upstream Regulatory Element (SURE) upstream of the TnIslow gene and a fast Intronic Regulatory Element (IRE) within the first intron of the TnIfast gene. Within the 144 bp IRE, there are 4 known cis elements, and the aim of this work was to initiate the studies to map the element(s) that are chiefly responsible for directing the fast-fiber-specificity of IRE-driven gene expression. This was approached by making IRE end-deletion constructs lacking either the left-most or right-most IRE cis-element. These IRE derivatives were coupled to a reporter gene consisting of a minimal (enhancer-dependent) TnIfast promoter linked to E. coli beta-galactosidase coding sequences. The transcriptional activity of these constructs was first evaluated in cell culture transfection experiments, and then by in vivo gene transfer into adult mouse skeletal muscles. The conclusion of these experiments was that fast-fiber-specificity of IRE-driven gene expression resides in the left-most 30 bp of the IRE, a region including an E-box binding site for myogenic regulatory factors of the MyoD family.

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I investigated SL trans-splicing in the troponin I gene of Ciona intestinalis. Experimental mutation of the AG dinucleotide adjacent to the natural trans-splice acceptor site (-64) in CiTnI/nuclacZ constructs eliminated trans-splicing to that site in Ciona embryos but activated trans-splicing at cryptic acceptor sites at -76 and -39, adjacent to the nearest AG dinucleotides. However, not all AG dinucleotides specify cryptic acceptor sites because outron internal deletions or 3'truncation mutants were trans-spliced at a far-upstream AG-adjacent cryptic site (-346), leaving many AGs in the retained outron segments. Thus, additional sequence elements that are present only in the -346 and -76/-64/-39 regions are required for cryptic acceptor activity. All mutant constructs generated detectable beta-gal enzyme expression, although the mutant with the longest retained-outron segment appeared less active. Therefore, mRNA accumulation and translation do not require trans-splicing to the natural acceptor site, although they may be facilitated by the normal removal of the outron during trans-splicing.

The fast-skeletal-muscle-fiber-specific expression of the troponin I(fast) (TnIfast) gene is driven by an Intronic Regulatory Element (IRE) located within the first intron of the gene. The IRE is a 148 bp transcriptional enhancer that contains several known and suspected cis-regulatory elements. These include the E-box, the closely-spaced MEF2 site and CACT box, the CACC site, and the CAGG element. Previous loss-of-function studies performed using the quail TnIfast IRE suggest that its activity depended on the MEF2 and CACT elements. The goal of my thesis research was to determine whether the MEF2 and CACT sites were not only necessary, but also sufficient, to support IRE activity. I prepared head-to-tail multimers of a 27-bp IRE segment that consisted largely of the near-adjacent MEF2 and CACT elements and did not contain any other known/suspected elements. These multimers were cloned upstream of a reporter gene consisting of the minimal promoter of the quail TnIfast gene linked to sequences encoding human placental alkaline phosphatase. The transcriptional capabilities of the constructs were assessed by gene transfer into the mouse soleus muscle in vivo by intramuscular injection/electroporation, and histochemical analysis of reporter enzyme plap expression including quantitative microdensitometry. I found that expression of these constructs was readily detectable and that it was markedly reduced by prior mutation of the CACT and, especially, of the MEF2 sites. These data indicate that the short DNA segment containing MEF2 and CACT elements is sufficient to drive expression in skeletal muscle and confirms the functional importance of these specific elements. / Although constructs containing the wild-type IRE 27-bp region were expressed, there was little preferential expression in fast fibers, in contrast to expression driven by the complete 148-bp IRE. Thus my results indicate that the MEF2 and CACT elements are not sufficient to drive fast fiber-type-specific expression, and suggest that additional elements outside of the 27-bp region tested are also necessary for fiber-type-specificity.

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