Tandem Affinity Purification of Myogenin and Characterization of the Tcap Promoter

Author

Shuang Zhang, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2010

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Davie, Judy

Abstract

Myogenesis is orchestrated by a family of four related bHLH transcription factors; MyoD, Myf5, Myogenin and Mrf4. The MRFs form heterodimers with E proteins and bind to the promoters or enhancers of muscle specific genes. Of these four, the Myogenic Regulatory Factor (MRF), myogenin, has been shown to play an important role during terminal differentiation of skeletal muscle. However, little is known about myogenin's function during myogenesis. In order to reveal myogenin associated factors, we performed tandem affinity purification of myogenin. The TAP purification with HEK293 cells elucidate that HEB and E2A are myogenin interacting factors. The expression of Tcap (Telethonin) is down regulated in myogenin null mice. However, beyond this, little is known about the regulation of the Tcap gene. In this work, we characterized the Tcap promoter by creating a luciferase reporter under the control of a minimal Tcap promoter that maintained the appropriate expression of Tcap in C2C12 myoblast cells. We performed luciferase assays with several Tcap reporter variants to characterize regulatory elements required for the expression of Tcap. Our results demonstrated that the Tcap promoter can be activated by exogenous myogenin or MyoD in NIH3T3 cells. In myoblasts, Tcap expression is greatly up regulated during differentiation, consistent with the expression of myogenin. Both of the two E boxes located at the Tcap promoter are required for the full Tcap activation in both NIH3T3 cells and C2C12 cells.