# Molecular analysis of negative and positive regulators of myogenesis

#### Abstract

The muscle regulatory factors (MRFs) MyoD, myogenin, Myf-5 and MRF4 are muscle-specific basic helix-loop-helix (bHLH) transcription factors which exhibit the unique capacity to initiate a myogenic developmental program when ectopically expressed in a variety of nonmuscle cell lineages. The commitment of myoblasts to terminal differentiation is controlled negatively by a variety of agents such as basic fibroblast growth factor (FGF-2), transforming growth factor $\beta1$ (TGF-$\beta1)$ and the activated Ras oncogene (Ras $\rm p21\sp{val}).$ The differentiation process is also positively regulated by the muscle LIM protein (MLP). MLP has two LIM motifs, each containing two zinc finger structures that are involved in protein-protein interactions. The mechanisms underlying the negative and positive regulation were investigated. To study whether Ras $\rm p21\sp{val}$ directly regulates the activity of the MRFs, we examined the DNA binding and transcription potentials of MRF4 and MyoD in cells expressing Ras $\rm p21\sp{val}.$ Our results demonstrate that Ras $\rm p21\sp{val}$ inhibits terminal differentiation by targeting the conserved basic domain of the MRFs, yet, the DNA binding and the transcriptional activity of the MRFs are not repressed by Ras $\rm p21\sp{val}.$ On the contrary, the transcriptional activity of the MRFs is repressed by FGF-2 and TGF-$\beta1.$ Therefore, Ras $\rm p21\sp{val}$ blocks myogenesis by an intracellular signaling pathway that is distinct from the bFGF and TGF-$\beta1$ pathways. In contrast to FGF-2 and Ras $\rm p21\sp{val},$ MLP positively regulates muscle differentiation. My results demonstrate that MLP enhances myogenic activity of the MRFs by a direct physical interaction, and this interaction is mediated by the first LIM motif of MLP and the bHLH domain of the MRFs. However, MLP is not a traditional transcription factor because it lacks a transcription activation domain. Nonetheless, MLP may act as a cofactor for the myogenic bHLH proteins by interacting with the basic domain of the MRFs and by enhancing the DNA binding ability of the MRFs. Together, these negative and positive regulators represent key components within the complex muscle regulatory networks which ensure that the process of myogenesis proceeds in an orderly fashion.

#### Degree

Ph.D.

Konieczny, Purdue University.

#### Subject Area

Molecular biology

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