Ken Hastings
More about mammalian TnI gene regulation research
Our gene regulatory studies in the higher vertebrates have focused on the TnIfast gene. Several lines of research have established that the TnIfast gene contains an important enhancer in the first intron (within the 5'-untranslated sequence). This enhancer, termed the IRE, is a ~150 bp element that was shown by our colleague at Purdue University, Steve Konieczny, to activate TnIfast gene expression during the in vitro differentiation of myoblasts in culture (myoblasts are the committed, but undifferentiated, embryonic precursor cells that will form muscle). We have studied TnIfast gene regulation in transgenic mice using constructs based on a bird (quail) TnIfast gene and have found that the IRE contains all the information required to direct skeletal muscle-specific, and fast skeletal muscle fiber-type-specific, gene expression. Thus the secret to the differential expression of the TnIfast gene lies within the 150 bp IRE enhancer. Several different cis-regulatory elements are present within the IRE, including binding sites for myoD-family, and MEF2-family muscle transcription factors, as well as binding sites for other, as yet uncharacterized trans-factors. We are currently producing mutant variants of the IRE, affecting one or more of the cis-elements, in various combinations, and testing the variants in transgeneic mice in an attempt to identify the cis-element and trans-factor that is chiefly respondsible for the fast fiber-type-specificity of the TnIfast IRE. Identification of the relevant cis element(s) and trans factor(s) will provide an important route into elucidating the cellular and molecular developmental mechanisms that direct muscle differentiation.
Transgenic mouse embryos expressing various beta-gal reporter transgene constructs based on the TnIfast gene IRE enhancer.
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