Muscle-specific and fiber type-specific regulation of the gene encoding the human slow-twitch skeletal muscle-specific isoform of troponin I

Abstract

Muscle-specific contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast versus slow twitch myofibers. This fiber type-specific gene regulation occurs by unknown mechanisms, and is not observed within cultured myocytes. Hence, the only means by which to study fiber type-specific gene regulation has been by generating numerous lines of transgenic mice, which is expensive and laborious. The goal of these studies was to develop an improved system by which the molecular mechanisms of fiber type-specific gene regulation could be dissected. The gene encoding the human slow twitch skeletal muscle-specific isoform of troponin I (TnI{dollar}\sb{lcub}\rm s{rcub}{dollar}) was chosen as a model gene, because expression of TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} is largely restricted to slow twitch myofibers in adult mammals. Structural analysis showed that the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} gene contains nine exons spanning 12.5 kilobases. Transcriptional analysis revealed two transcription initiation sites. A muscle-specific promoter and a muscle-specific enhancer were identified 5' to the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} transcription initiation region. Next, an assay by which to identify DNA elements involved in fiber type-specific gene regulation was developed: this assay employs gene transfer into the muscles of live rats. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow versus fast twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well-expressed by both muscle types when fused to a non-fiber type-specific skeletal actin promoter. Deletion analysis of the TnI{dollar}\sb{lcub}\rm s{rcub}{dollar} promoter region revealed that the 157 base pair muscle-specific enhancer conferred slow muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow twitch myofibers. This delineation of a fiber type-specific control element represents a significant advance toward understanding fiber type-specific gene regulation at the molecular level. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber type-specific gene expression, prior to the generation of transgenic mice.