NCI Funded Research Portfolio - Z01 BC 005258 Abstract

Z01 BC 005258 (Z01)
Title	Molecular Studies of Eukaryotic Gene Regulation
Institution	NCI, Bethesda, MD
Principal Investigator	Paterson, Bruce	NCI Program Director	N/A
Cancer Activity	N/A	Division	CCR
Funded Amount	$659,462	Project Dates	10/01/1980 - N/A
Fiscal Year	2007	Project Type	Intramural
Research Topics (SICs) w/ Percent Relevance		Cancer Types (Disease Sites) w/ Percent Relevance
Molecular Biology (100.0%)		N/A
Common Scientific Outline
Application of Model Systems Normal Functioning
Abstract	We have made substantial progress in understanding the role of nautilus in myogenesis in the fly embryo. The highly organized and segmentally reiterated muscle pattern in the Drosophila embryo is prefigured by the arrangement of a sub-population of mesodermal cells called founder myoblasts. We had shown earlier that the expression of nautilus, the only MyoD-related gene in Drosophila, is initiated at stage 9 in a stereo-specific pattern in a subset of mesodermal cells that become incorporated into every somatic muscle in the embryo. Targeted ricin toxin ablation of these cells resulted in the loss of embryonic muscle. We now know that at stage 11 these same cells begin to express the founder cell-specific marker, duf LacZ (rP298LacZ) thus nautilus is the earliest marker for the critical founder myoblast population. We inactivated the nautilus gene using homology-directed gene targeting and a novel gal4-inducible nautilus RNAi transgene to determine if any aspect of founder cell function required nautilus gene activity. An earlier study using the injection of nautilus dsRNA to induce gene silencing by RNAi indicated loss of nautilus function would result in a severe embryonic muscle loss or disruption. Both gene targeting and gal4-inducable RNAi resulted in a range of defects that included severe embryonic muscle disruption, reduced viability and female sterility, all of which were rescued by a nautilus transgene. More importantly, the highly organized founder cell pattern that is needed to establish the proper embryonic muscle organization was disrupted in nautilus null embryos prior to MHC expression and prefigured the subsequent embryonic muscle defects observed at later stages in development. Tinman, a marker for mesodermal cells that give rise to the dorsal vessel or heart, was expressed normally in the nautilus null. Although nautilus does not specify the myogenic cell lineage, it has a cell autonomous role in establishing the correct muscle organization in the embryo through its regulation of the founder cell pattern. This work has been published recently in PNAS. We are currently carrying out experiments to identify nautilus target genes that are likely important for cell-cell recognition, cell movement and migration-issues important to understanding the regulation of normal cell growth and patterning in human disease and development. To identify nautilus target genes we have used two approaches. First we have undertaken a transcriptome analysis of mutant and wild-type embryos using the Solexa 1G Genomic Analyzer, a so-called deep sequence approach. This should reveal differences in the abundance and classes of RNAs dependent upon nautilus function. Second we have engineered two types of tags on the engodenous nautilus gene, one a V5-Flag fusion and the other a peptide sequence that can be biotinylated by E. coli biotin ligase expressed from a gal4-inducible transgene. This will enable us to perform ChIP-Seq to directly sequence nautilus target DNA sequences immunoprecipitated either with V5-Flag antibodies or avidin tagged antibody, again using the Solexa 1G Genomic Analyzer to determine nautilus target elements. Target gene candidates will be analyzed in vivo using available mutant stocks or inducible RNAi stocks made in the lab or obtained from the Vienna Drosophila RNAi Center. We have recently identified a set of novel miRNAs that are candidates for the post transcriptional regulation of nautilus in the embryo. The miRNA binding sites are found in the 3'UTR of the nautilus gene in four species of Drosophila and the nautilus 3'UTR can regulate reporter expression in response to the ectopic expression of the miRNAs in S2 cells. We have engineered flies that express these miRNAs under gal4 control in numerous cell types including founder cells. We have also introduced a nautilus transgene without the miRNA target sequences as a rescue construct in the mutant background to see if misexpression of nautilus in the absence in miRNA regulation affects myogenesis. These reagents are currently under investigation with results expected in the near future. To gain insight into the molecular basis of RNAi-induced gene silencing, we identified a novel mechanism in Drosophila that appeared to involve an RNA-dependent RNA polymerase (RdRP) activity in RNA target degradation. siRNAs, produced by the Dicer RNase III-related enzymes in response to the trigger dsRNA, were shown to act as primers to convert the target mRNA into new dsRNA which was then degraded again by Dicers in a cycle of amplification and degradation. This was termed degradative PCR. This was the first biochemical evidence to shed light on the role of the siRNAs in RNAi and provided a basis to explain the potentcy of the mechanism in post transcriptional gene silencing since very few molecules of dsRNA were able to inactivate hundreds of target mRNA molecules. RdRP is a highly conserved key component in RNAi in C. elegans and lower eukaryotes and plays a role in heterochromatin maintenance as well. We have now enriched the RdRP activity sufficiently to obtain mass spec data and this has allowed us to identify the the protein, the first example of a highly conserved noncanonical RdRP in eukaryotes (S. pombe to humans) involved in RNAi. The RdRP is both primer dependent and independent and interacts with other key components of the RNAi machinery. A manuscript describing this important find is in preparation. This noncanonical RdRP may be important not only for RNAi but also for heterchromatin formation and maintenance since a fraction of the RdRP is nuclear and loss of the protein is lethal in Drosophila. This relationship is under investigation. In our RNAi studies we also cloned Drosophila Dicers 1 and 2 and expressed the full-length cDNAs in baculovirus to produce the active enzymes. Each is an RNase III with different properties that place Dicer 2 with the RNAi pathway and Dicer 1 in the miRNA pahtway. We have also identified novel RNA-binding proteins that regulte the activity of the Dicers in these pathways. We are exploring the interaction of the Dicers with RdRP and the targeted degradation of mRNA. Preliminary results suggest there is an Ago-dependent pathway involving siRNA-directed cleavage of the target through Ago, and an RdRP-dependent pathway involving siRNA priming and Dicer cleavage in a repeatative cycle to degrade the mRNA.