The Regulation of MyoD, Myogenin, Id2, Id3 and RP58 Gene Expression in MyoD Knockdown Cells Throughout Myogenesis

Graduate #20
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Kumar Tiger - California State University Los Angeles

Skeletal muscle development is essential in developing organisms because muscle is important for protection, breathing, and locomotion. Myogenic differentiation factor (MyoD) and myogenic factor 5 (Myf5) are closely related transcriptional regulatory factors that are well known to positively regulate the expression of muscle specific genes during differentiation. Currently it is unexplained how and to what extent MyoD or Myf5 regulates the genes for the inhibitors of DNA binding and differentiation (Id). MyoD binds to the regulatory regions of the Id2 and Id3 genes during proliferation and early differentiation, but the function of the bound MyoD is unknown. One possibility is that MyoD contributes to efficiency of Id gene transcription. During differentiation the upregulation of Repressor Protein 58 (RP58) by MyoD leads to downregulation of transcription of the Id2 and Id3 genes. Also, forced expression of Id proteins inhibits differentiation by preventing formation of the MyoD/E protein heterodimer that activates muscle-specific genes. Our hypothesis is that MyoD contributes to the expression of its own inhibitor during proliferation and/or the early stages of myogenesis. Previously, stable transgenic C2C12 myoblast cell lines were made with MyoD knockdown constructs expressing shRNAs. These knockdowns were generated for the purpose of determining whether the presence of MyoD affects Id2 and Id3 mRNA levels during proliferation and early differentiation. We are using qRT-PCR and Western blot to identify which of the transgenic lines are successful knockdowns. Our recent results provide a tentative identification of MyoD knockdown lines based on reduced levels of MyoD and myogenin mRNA. The effect of loss of MyoD on levels of Id2, Id3, and RP58 mRNAs will be determined with a qRT-PCR over a time course of proliferation and differentiation, comparing the results from MyoD knockdown cell lines with those from wild-type C2C12 myoblast cell lines and control non-target shRNA knockdown cell lines.

Not Submitted

Funder Acknowledgement(s): CSU LSAMP-BD Cohort XI is supported by the NSF via Grant # HRD-1363399

Faculty Advisor: Sandra Sharp, ssharp@exchange.calstatela.edu