| The withdrawal of growth factors from proliferating myoblasts leads to the induced expression of muscle specific genes essential for myogenesis. Using a suppression subtractive hybridization technique (SSH), we have cloned a novel human cDNA that encodes a Cys3His zinc finger protein named CHCR (C&barbelow;s3H&barbelow;is C&barbelow;CG1-R&barbelow;equired). CHCR is related to Muscleblind (Mbl), a Drosophila melanogaster protein required for terminal muscle differentiation. It also displays 60% amino acid identity to EXP/MBNL, a human Mbl protein that interacts with CUG expansions associated with the degenerative muscular disease, myotonic dystrophy (DM1). This relationship with EXP/MBNL and Mbl suggests that CHCR also functions during muscle differentiation. We have found that CHCR mRNA levels decrease upon differentiation of mouse MM14 and C2C12 myoblast cells. Constitutive expression of CHCR in C2C12 cells inhibits the induction of sarcomeric myosin heavy chain (MyHC) upon serum deprivation. Induction of myogenin, an earlier marker of muscle differentiation, is inhibited to a lesser extent, while expression of the cell cycle inhibitor, p21, remains unaffected. Loss of CHCR function by treatment with morpholino antisense oligonucleotides accelerates MyHC induction during differentiation of myoblast cells. These complementary gain and loss of function results suggest that CHCR is an inhibitor of myogenesis. CHCR represents the first muscleblind-related protein that antagonizes, instead of promotes, the process of muscle differentiation.; In this thesis, we also characterize the genomic structure, chromosomal localization, and isoform variation of the human CHCR gene. The human gene is located on the X chromosome at position Xq26.1, and is composed of 9 exons dispersed over 70 kbp of the genome. All of the intron acceptor and donor sites for exon splicing conform to the consensus GT/AG rule for eukaryotic genes. We have identified three splice variant isoforms of the human protein ranging in size from 334 to 354 amino acids. Our characterization of the CHCR gene and protein isoform variants provides a framework for future analysis of CHCR's role in regulating myogenesis in humans.; We have cloned a mouse CHCR cDNA containing the entire ORF by a homology-based PCR approach and analyzed its expression in adult tissues. The mouse CHCR protein is 342 amino acids in length and exhibits approximately 80% identity with the three human isoforms. RT-PCR analysis revealed that the mouse CHCR transcript is enriched in the lung, spleen, and testis of adult mice, but not in the heart and skeletal muscle. Since there is 80% sequence identity between the mouse and human CHCR proteins, cloning of the mouse CHCR protein and characterization of mRNA expression patterns in adult tissues will further our understanding of the conserved role of CHCR in mammalian differentiation.; Finally, we provide evidence that CHCR is a nuclear protein that exhibits a binding preference for DNA over RNA. It appears that the Cys3His motifs of CHCR are required to mediate this DNA interaction. The Cys3His motifs are >90% conserved between members of the Muscleblind family suggesting a functional relevance for this motif. Although these results are preliminary, they suggest a number of interesting future studies. |
