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 MRF regulation of the phosphorylation status of the retinoblastoma protein, Rb. (Project 1) We have recently shown that 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 as a fusion protein with GST or GFP inhibits the kinase activity of cdk4 and 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. Cdk4 affinities for the various binding domain derivatives are being determined uisng the BiaCore. Recently we have also determined the 15 amino acid domain binds to the other major G1 cyclin-dependent kinases, cdk6 and cdk2. We are determining whether or not the domain also blocks the corresponding kinase activity. Thus 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. This 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, induced antisense expression to nautilus RNA, or injection of double stranded nautilus RNA into the embryo (RNA interference or RNA-i) all eliminate 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. RNA-i does not appear to work in mammalian cells, as yet, even if we block PKR activity and the induction of apoptosis through the interferon pathway by using PKR -/- cells, the PKR inhibitor, p58, and the apoptotic inhibitor, p35, from baculovirus in various combinations. Embryonal stem cells (ES cells) are also very susceptible to killing by dsRNA so developmental stage does not appear to be a key factor, except in the case of the pre-implantation mouse embryo. In an effort to understand the molecular basis of RNAi in Drosophila for its eventual application to mammalian systems, we have recently uncovered a novel mechanism we have termed """"""""degradtive PCR"""""""" involving RNA-dependent RNA polymerase (RdRP) and the 21-25 nucleotide RNAs produced from the trigger dsRNA, called siRNAs. The short RNAs serve as primers to convert the target RNA into dsRNA which is then degraded by RNase III activity 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 purifying the RdRP from Drosophila. (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 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 established 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. 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.
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