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) provide excellent model systems 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. Furthermore, greater than 90% of the genes expressed in the dividing muscle cell are shut down in a process that involves massive chromatin reorganization while the muscle-specific genes are activated. 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 also determined that the MyoD 15 amino acid domain 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 purified 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 eventhough MyoD is a nuclear protein. Mouse fibroblasts from cdk4 mutant mice are available (S. Rane NIH) that either have no ckd4 or have a cdk4 that does not bind p16, the cdk inhibitor. We would like to use these mutants to check if the MyoD inhibitory domain binds to the same regions as p16 and to see if myogenic conversion is enhanced due to a failure to regulate the MyoD-cdk4 interaction. Myogenic conversion by MyoD may be enhanced in cdk4-/- cells. 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 of gene function. However, this method is frought with injection artifacts and must be controlled carefully. As a more practical approach,we have been trying to develop a Drosophila vector system that allows the induction of dsRNA in selective tissues at particular times during development. Our first attempts using an inducible T7 system worked in cultured S2 cells but turned out to be lethal when activated in the fly due presumably to cryptic T7 binding sites in the genome. A second approach, using the gal4 system and a flp-induced hairpin structure, demonstrated targeting of eye color with a white hairpin but screening for the flp induced hairpin makes the method less convenient. A third approach that allows the direct cloning of inverted repeats either side of an intron under the control of gal4 looks very promising and has proven successful in cultured S2 cells in the targeting of GFP. This is being tested in flies using white and nautilus (MyoD) since there is some contraversy regarding the nautilus null phenotype. We have recently started to use the newly developed gene targeting method (developed by Dr. Yikang Rong, NCI) to knock out genes involved in Drosophila myogenesis and RNAi(see below. As a first test of the method we have successfully targeted the inactivation of the nautilus gene by inserting the GFP gene under the control of the armidillo promoter in the middle of the nautilus gene by ends-out homologous recombination. The mutant is not lethal in the F1 generation but is lethal in the F2 generation. This is under analysis. Both the hairpin and direct targeting method will be used to study the role of genes important for RNAi. In efforts to understand the molecular basis of RNAi in Drosophila and higher eukaryotes we have reported on 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 enzymes, called the Dicers, to produce new primers while degrading the target RNA in the process. This result sheds light on the role of the siRNAs in RNAi and may explain the potentcy of the mechanism behind RNAi and post transcriptional gene silencing since very few molecules of dsRNA can inactivate 100-fold more of the cognate RNA. We are in the process of purifying the RdRP from Drosophila. We have also cloned Drosophila Dicers 1 and 2 and expressed the full-length cDNAs in baculovirus to produce active enzymes.
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