Tissue formation during development involves the determination, controlled proliferation and specific differentiation of cells in the embryo. Misregulation in any phase of this process can lead to failure in the development of the embryo, severe disease or uncontrolled cellular growth. Thus the study of gene regulation during development provides insight into areas important in human disease. Embryonic muscle formation in vertebrates and Drosophila (the fruit fly) provides an excellent model system in which to study the origin of one of the major tissues in higher organisms. The determination, proliferation, and differentiation of muscle cells during development in both vertebrates and invertebrates depend upon the function of the MyoD family of basic helix-loop-helix proteins, the muscle regulatory factors (MRFs). Determination of the first muscle precursor cells involves the activation of the MRFs in early mesoderm while gene expression characteristic of differentiated muscle remains repressed. Terminal differentiation is marked by the withdrawal of the myoblast from the cell cycle just prior to the activation of the muscle-specific genes and both processes involve the MRFs. Cell cycle control during terminal differentiation is thought to involve the MRFs in a pathway that regulates the phosphorylation status of the retinoblastoma protein, Rb. (Project 1) We have recently shown that ectopically expressed MyoD binds directly to the G1 cyclin-dependent kinase cdk4 to inhibit cell growth and the phosphorylation of Rb. The cdk4-MyoD interaction also blocks the trans activation functions of MyoD by disrupting DNA-binding by the MyoD/E-protein heterodimer. Therefore, high levels of nuclear cdk4 block MyoD function in growing myoblasts while the loss of nuclear cdk4 in the absence of growth factors and mitogens allows MyoD to function. We have identified a 15 amino acid domain on MyoD responsible for the interaction with cdk4. Expression of this domain either as a fusion protein with GST or GFP inhibits the kinase activity of cdk4 in vitro and in vivo, blocking its ability to phosphorylate the retinoblastoma protein, Rb. This results in the cessation of cell growth and induces myoblast differentiation in the presence of mitogens. We have a patent application on the inhibitory activity of the 15 amino acid domain of MyoD on cdk4 kinase activity. We have recently made alanine substitutions in all the positions of the 15 amino acid cdk4-binding domain in order to map the critical residues for interaction. Single substitutions have a marginal affect on inhibitory and binding activity of the domain but two simultaneous substitutions reduce cdk4 binding and kinase inhibition for the various binding domain derivatives. The binding parameters are being determined uisng the BiaCore and the imobilized 15 amino acid derivatives. We have recently determined that the MyoD 15 amino acid domain also binds to the other major G1 cyclin-dependent kinases, cdk6 and cdk2. cdk6 behaves like cdk4 during muscle differentiation in that cdk6 leaves the nucleus when mitogen levels are reduced but can be induced to re-enter myotube nuclei with the expression of a stable cyclin D1 in the cells. cdk6 phosphorylation of Rb is also inhibited by the MyoD binding domain. However, although cdk2 binds to the same 15 amino acid domain, phosphorylation of histone in vitro is not inhibited. All the in vitro kinase assays are performed using baculovirus produced cyclin D1/cdk4, cyclin D1/cdk6, and cyclin E/cdk2 prufied by Flag-tag affinity chromatography. cdk4/6 kinases are inhibited by p16 and p21 while cdk2 activity is only blocked by p21. We suggest that in the dividing myoblast the G1 cdks can act to hold MyoD activity in check until the cell begins to exit the cell cycle as mitogen levels are lowered. Chromatin immunoprecipitation assays with MyoD antibody indicate MyoD is not associated with its target genes in the dividing myoblast yet the protein is nuclear. This MyoD interaction with cell cycle related proteins may be a more general mechanism with regard to other tissue-specific bHLH transcriptions factors and this is being examined with NeuroD2, a neurogenic bHLH protein. In Drosophila we have also shown that MyoD (nautilus) expression defines a subset of mesodermal cells that are required to set up the muscle pattern in each hemisegment of the embryo. Ricin toxin ablation of nautilus positive cells, or injection of double stranded nautilus RNA into the embryo (RNA interference or RNA-i) alter normal muscle formation in the embryo and define nautilus as an essential gene for myogenesis in the fly. This study demonstrated the general utility of RNA-i ablation of gene function in Drosophila in the absence of a genetic mutation and is the method of choice for a rapid analysis. We have developed a Drosophila vector system to induce dsRNA in selective tissues at particular times during development and this is under analysis. Preliminary results using nautilus as a test gene suggest loss of nautilus function is a lethal that results in disruption of the normal muscle pattern, similar to the results obtained by direct injection of the dsRNA. Control transgenic flies with the inducer gene alone that does not produce dsRNA are under study to confrim that the phenotype observed is due to the induction of dsRNA for the target gene. Unfortunately, similar treatment of mammalian cells with dsRNA larger that 30 nucleotides induces cell death via apoptosis. In an effort to understand the molecular basis of RNAi in Drosophila we have recently uncovered a novel mechanism we have termed degradtive PCR that appears to involve an RNA-dependent RNA polymerase (RdRP) and the 21-25 nucleotide RNAs produced from the trigger dsRNA, called siRNAs for short interfering RNAs. The short RNAs serve as primers to convert the target RNA into dsRNA which is then degraded by an RNase III-related enzyme, called Dicer, to produce new primers while degrading the target RNA in the process. This results explains the underlying mechanism behind RNAi and post transcriptional gene silencing. We are in the process of trying to identify the RdRP from Drosophila. We have also cloned Drosophila Dicer and expressed the full-length cDNA in baculovirus to produce active enzyme. We have identified other components of the RNAi system in Drosophila by gene comparison using genetic studies from C.elegans, Arabidopsis, and neurospora. Dicer as well as these associated proteins are being used as bait to characterize protein complexes capable of performing various steps in RNAi. (Project 2) Determination of the myoblast in the mesoderm involves the activation of MyoD and MyoD responsive downstream target genes. We have been studying the activation of the single MyoD gene, nautilus, in Drosophila and have determined that the nautilus promoter is activated predominately by DMEF2, a Drosophila SRF homolog, and twist, a major determinant of the mesoderm. We can induce Schneider cells to activate a partial myogenic program by expressing daughterless in these cells. This myogenic conversion is potentiated by the co-expression of DMEF2 and nautilus. The cells exit the cell cycle, become multinucleated and express muscle-specific myosin similar to embryonic muscle. Myogenic conversion of Schneider cells by daughterless is dependent upon the endogenous expression of very low levels of nautilus and DMEF2, two markers that establish Schneider cells are of mesodermal origin. Inhibition of the endogenous gene function for nautilus and DMEF2 by RNA interference established these gene products were essential for myogenic conversion of Schneider cells by daughterless. Quantitative RT-PCR has shown that Schneider cells express 100-1000 fold less daughterless than nautilus. Raising the levels of daughterless protein by ectopic expression allow sufficient levels of the nautilus/daughterless heterodimer to form to activate the myogenic program. This work defined conditions for the application of RNAi to cultured Drosophila cells and established a myogenic system in which to analyze nautilus-responsive genes by micro array analysis. We have established a daughterless inducible KC cell line using the metallothionine promoter system which converts to a muscle phenotype upon induction. This system is being used to identify nautilus target genes by microarray. RNA interference is being used to explore the role of other factors in the myogenic process as well. We hope to analyze genes that are differentially expressed between the non-myogenic and myogenic state to identify early target genes for myogenic conversion. We have recently begun to culture primary embryonic Drosophila muscle and hope to use RNAi to explore the role of genes identified in the microarray studies on the differentiation of the primary cultures.
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