The long-term goal of this research is to unravel the molecular mechanism of muscle differentiation and to provide new information that may lead to therapeutic approaches for myotonic dystrophy. The activation of muscle differentiation involves both positive and negative signals. While much attention has been placed on proteins that promote muscle differentiation, the importance of negative regulatory pathways has been under emphasized. Disrupting the delicate balance between positive and negative signals can lead to the development of disease. The focus of this proposal will be to investigate the function of a novel protein CHCR that we hypothesize antagonizes muscle differentiation, which is in striking contrast to its close relative Muscleblind (at 49% identity), a Drosophila protein essential for muscle formation. CHCR also displays approximately 80% homology to proteins implicated in myotonic dystrophy (DM l) and binds to expanded repeat transcripts found in the nuclei of DM1 cells. Based on our preliminary studies, we hypothesize that CHCR is an inhibitor of muscle differentiation and that its mechanism of action requires changes in gene expression mediated by direct DNA binding. To test this hypothesis, we will examine the effects of CHCR overexpression and reducing CHCR protein levels by morpholino antisense oligonucleotides and RNA interference on muscle differentiation in C2C12 mouse myoblasts. We also propose to map the part of CHCR that is required for suppression of muscle differentiation. This information will help guide the protein interaction studies described below. The techniques of suppression subtractive hybridization and DNA microarrays will be used to identify changes in gene expression mediated by CHCR function. The involvement of direct DNA binding will be investigated by chromatin immunoprecipitation followed by the probing of DNA microarrays (ChiP-chip) and a random site selection technique. A yeast two-hybrid screen and coimmunoprecipitation experiments will be carried out to identify CHCR-binding proteins required for its antagonistic effect on mammalian muscle differentiation. The identification of direct gene targets and CHCR interacting proteins will provide the tools to further investigate CHCR function in muscle differentiation. Our long-term goal is increase our understanding of negative regulatory mechanisms underlying mammalian muscle differentiation. Such findings may reveal new avenues for therapy development.
| Lee, Kyung-Soon; Cao, Yi; Witwicka, Hanna E et al. (2010) RNA-binding protein Muscleblind-like 3 (MBNL3) disrupts myocyte enhancer factor 2 (Mef2) {beta}-exon splicing. J Biol Chem 285:33779-87 |
| Lee, Kyung-Soon; Smith, Kimberly; Amieux, Paul S et al. (2008) MBNL3/CHCR prevents myogenic differentiation by inhibiting MyoD-dependent gene transcription. Differentiation 76:299-309 |
| Lee, Kyung-Soon; Squillace, Rachel M; Wang, Edith H (2007) Expression pattern of muscleblind-like proteins differs in differentiating myoblasts. Biochem Biophys Res Commun 361:151-5 |
| Bender, Andrew T; Ostenson, Cari L; Wang, Edith H et al. (2005) Selective up-regulation of PDE1B2 upon monocyte-to-macrophage differentiation. Proc Natl Acad Sci U S A 102:497-502 |
