Troponin I gene regulation and the role of the muscle regulatory factors in muscle differentiation

Katherine Eugenia Yutzey, Purdue University

Abstract

During skeletal muscle development, approximately thirty genes encoding the contractile proteins and related gene products are transcriptionally activated as myoblasts differentiate into myofibers. One of these genes, the fast isoform of skeletal troponin I (TnI), is regulated primarily by an internal regulatory element (IRE) located in the first intron of the gene. The 148 bp IRE is a muscle-specific enhancer that contains three specific protein binding sites, each of which is necessary for TnI gene activation. The most 5$\sp\prime$ of these binding regions contains a bHLH protein binding site consensus, or E-box, that has been shown to bind the muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4. Sequences similar to the remaining sites are present in the regulatory elements of other contractile protein genes, but the transcription factors that bind these sites are uncharacterized. The three regions or sub-elements are interdependent, since mutation of any one site results in $>$80% loss in enhancer activity. Although MyoD, myogenin, Myf-5, and MRF4 each are involved in muscle determination and differentiation, the distinct roles of these proteins are unknown. A direct comparison of the abilities of these proteins to trans-activate the expression of contractile protein genes revealed that MyoD and myogenin are potent trans-activators of all genes tested. MRF4, however, was unable to trans-activate muscle-specific expression of any gene with the exception of cardiac $\alpha$-actin. Transcriptional activation by each muscle regulatory factor is inhibited by basic fibroblast growth factor via a signal transduction pathway that may affect the muscle regulatory factors post-translationally. The ability of the muscle regulatory factors to activate contractile protein gene expression is dependent on the sequence of the E-box binding site. Replacement of the TnI IRE E-box site with the corresponding E-box sites from other contractile protein genes significantly affects the ability of MyoD to activate TnI gene transcription. Thus specific interactions between distinct contractile protein gene regulatory elements and a variety of transcription factors, including the muscle regulatory factors, govern transcriptional activation during muscle differentiation.

Degree

Ph.D.

Advisors

Konieczny, Purdue University.

Subject Area

Molecular biology

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