Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Simon H. Chang

Abstract

The rabbit muscle phosphofructokinase (RM-PFK) gene is predominantly expressed in skeletal muscle. Its muscle-specific transcription was investigated using transient transfection studies, electrophoretic mobility shift assays, and DNaseI footprinting experiments. The evidence presented here provides the first example of the myogenic basic-Helix-Loop-Helix proteins regulating a gene that encodes a glycolytic enzyme. The 3 kb 5$\sp\prime$-flanking sequence of this gene directed the expression of the chloramphenicol acetyltransferase gene in muscle and non-muscle cells. MyoD, Myf5, and myogenin exhibited differential transactivation capabilities. MyoD had the most pronounced transactivational effect, and its N-terminal transactivation domain and the basic DNA-binding region were required for its function. The RM-PFK gene has at least two muscle-specific promoters that are approximately 1.9 kb apart, and that were stimulated by the myogenic regulators to different extents. The proximal promoter was the major promoter responding to transactivation of the myogenic regulators. Progressive deletional analysis revealed that there were two positive and two negative regulatory regions controlling the proximal promoter. An enhancer region, E1, contains a CAGCTG E-box that was recognized by MyoD glutathione S-transferase fusion in vitro. Along with this E-box, two other specific sequences were also recognized by the nuclear proteins of C2C12 cells. The TATA-less proximal promoter was localized to a 268 bp region, with its core promoter lying within region $-$95 to +23 with respect to the cap site. A pyrimidine-rich initiator and a myotube-specific AG-rich sequence within the core promoter were recognized by the nuclear proteins of C2C12 cells. MyoD stimulation was mediated through the sequences around the core promoter, mainly through the region ($-$195 to $-$95) containing a crucial CAGATG E-box. A CAGCTG E-box and a CCATCGT sequence downstream of the core promoter were also recognized by the nuclear proteins of C2C12 cells. In contrast, the distal promoter b was localized within a 671 bp region and might contain a TATA element (TTATTTATT). Thus, the differences in regulation of these promoters may be correlated with their divergent DNA compositions.

Pages

168

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