• Developmental regulation of the human slow troponin I isoform gene in skeletal and cardiac muscles

      Zhu, Lei; Wade, Robert P. (1996)
      The differentiation and maturation of striated muscle involves numerous alterations in the pattern of contractile protein isoform gene expression which ultimately lead to the mature phenotype of distinct muscles. The molecular mechanisms controlling these processes remain poorly understood. Troponin I (TnI) is one of the contractile proteins that is subjected to this complex regulation. Three TnI isoform genes are differentially expressed in slow and fast skeletal myofibers and adult cardiac muscle. We first studied the expression profile of the three TnI isoform genes during murine development. Detailed analysis showed that the slow isoform of TnI (TnI{dollar}\rm \sb{lcub}S{rcub}){dollar} is the predominant isoform found in early development of both skeletal and cardiac muscles. The differential expression pattern of TnI isoform genes occurs at late fetal and postnatal stages. During this period, TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} becomes restricted to slow twitch skeletal myofibers and the conductive tissues of the heart. Next, we focused on the regulation of the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene, in an effort to identify cis-regulatory elements which govern its skeletal muscle fiber type-specific gene expression as well as those regulating TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene expression in the developing heart. Transgenic mice harboring 4,200 bp of the 5{dollar}\sp\prime{dollar} flanking sequence of the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene exhibited proper transgene expression in adult slow twitch myofibers and in the developing heart with the notable exception of aberrantly low level of expression in fetal ventricles. In vitro transfection assays had identified two enhancer elements, an upstream USE and an intron 1 INE, and sequence within the first 95 bp of upstream from the transcription start site which play important roles in the cell type-specific expression. Additional lines of transgenic mice driven by various combinations of the cis-regulatory elements from the TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene were generated to further define their in vivo functions. Our results suggested that separate regulatory elements regulate the human TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene in skeletal versus developing cardiac muscles. The USE or INE is able to activate the {dollar}-{dollar}95 promoter in the developing skeletal muscle. In addition, activity of the INE is down-regulated during postnatal development and it appears not to be involved in slow skeletal muscle fiber type-specificity. The differences between the activities of these two enhancer elements support our hypothesis that the INE may be required for proper developmental regulation, and the USE is the primary determinant for slow fiber restricted gene expression. However, in contrast to in vitro expression data, even the combination of the two enhancers is not sufficient to activate the {dollar}-{dollar}95 promoter in cardiac muscle. Our studies show that aspects of the spatial and temporal expression pattern of a tissue-specific gene during development can be conferred by separate and distinct regulatory elements. Moreover, expression of the TnI{dollar}\rm \sb{lcub}S{rcub}{dollar} gene in the embryonic and fetal heart may be governed by a complex mechanisms some of which are not readily amenable to in vitro study.
    • Muscle-specific and fiber type-specific regulation of the gene encoding the human slow-twitch skeletal muscle-specific isoform of troponin I

      Corin, Shari Jill; Wade, Robert P. (1995)
      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.