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Developmental regulation of the human slow troponin I isoform gene in skeletal and cardiac muscles
Abstract
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.Description
University of Maryland, Baltimore. Molecular and Cell Biology. Ph.D. 1996Keyword
Biology, MolecularBiology, Cell
Health Sciences, Human Development
Contractile Proteins
Gene Expression Regulation, Developmental
Troponin I--genetics