David Gilmour - US grants

DESCRIPTION (provided by applicant): This submission is in response to Notice Number NOT-OD-09-058 entitled: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The development of massive parallel sequencing platforms opens new avenues of discovery that could significantly expand the scope of our work. We plan to develop a method for mapping paused Pol II across the entire Drosophila genome by combining permanganate genomic footprinting, chromatin immunoprecipitation, and massive parallel sequencing. Several recent studies have shown that Pol II is concentrated in the promoter proximal region of many genes and raise the possibility that this is a prominent regulatory step in gene expression. However, most studies have relied on chromatin immunoprecipitation, which is unable to determine if Pol II is transcriptionally engaged. Our method will provide a high resolution measure of the distribution of paused Pol II. We also want to combine ChIP and massive parallel sequencing to determine the distribution of a novel sequence specific DNA binding protein that our evidence indicates could be involved in promoter proximal pausing on 30% of the genes we predict have paused Pol II. PUBLIC HEALTH RELEVANCE: Control of gene expression is essential for normal development, and many diseases can be attributed to the miss-regulation of genes. Gene expression is usually controlled at the level of transcription. This research investigates mechanisms of transcriptional control using Drosophila as a model system for understanding the process in humans because Drosophila is amenable to genetic, molecular genetic and biochemical analyses.

Gilmour 9723537 A new approach for investigating the function of transcriptional activators in living cells will be developed. A collection of 6 factors and RNA polymerase II compose the general machinery that is required for initiation of transcription but the activity of the general machinery is regulated by DNA sequence-specific binding proteins called activators. Genomic fingerprinting provides a way to monitor protein-DNA interactions in living cells and recently a new method using permanganate has been developed in this laboratory to monitor the interactions of TFIID and RNA polymerase II on promoters that have been transformed into Drosophila. Permanganate footprinting of a transformed promoter can be done in intact salivary glands that have been dissected from larvae, providing a true picture of an in vivo situation. The specific aims of this project are: 1) to use available fly stocks to determine if a GAL4 based approach can be used to investigate how an activator affects the promoter interactions of TFIID and RNA polymerase II in living cells 2)to determine how Gal4-VP16 and Gal4(10147) affect target promoters carrying various numbers of Gal4-binding sites 3) determine what changes occur in promoter interactions when the activator is switched on or off by using derivatives of the Tet-repressor. New insights into determining mechanisms for the regulation of transcription in living cells will result from this work. Although tremendous progress has been made in the identification and biochemical characterization of factors involved in transcriptional regulation of gene expression, much of the work has been done using artificially contrived systems or by indirect analysis. The ability to analyze such interactions in living cells should be useful in obtaining a more complete understanding of how gene expression is regulated.

Genes are composed of DNA and contain the information encoding proteins in cells. In eukaryotes, this information is transcribed from DNA into RNA by an enzyme called RNA polymerase II. Much is known about how RNA polymerase II associates with the beginning of a gene to initiate transcription but little is known about how RNA polymerase II disengages from the DNA after reaching the end of a gene. This disengagement is known as termination. This project investigates the mechanism by which RNA polymerase II terminates transcription at the end of a gene. The protein, Pcf11, was found to disengage RNA polymerase II from DNA by a mechanism in which Pcf11 forms a bridge between the RNA being synthesized and a large protrusion on the RNA polymerase II known as the CTD. To elucidate the mechanism of this reaction, the effect of purified Pcf11 on purified RNA polymerase II engaged in transcription will be analyzed. Pcf11 was also found to associate with the end of a heat shock gene in live Drosophila cells, and depletion of Pcf11 from the cells inhibited the ability of RNA polymerase II to disengage from the heat shock gene. To determine how broadly Pcf11 functions in termination, this project will investigate the role of Pcf11 in causing termination on numerous other genes in Drosophila cells. Finally, specific sequences in the transcript are known to contribute to the termination mechanism. In contrast, isolated Pcf11 appears to cause termination indiscriminately. To determine the basis for the sequence specificity of termination, the ability of Pcf11 in association with other cellular factors to cause termination will be investigated. In addition, another protein from yeast called Nrd1 will be evaluated for termination activity, since this protein is predicted to have the ability to bridge the CTD to the RNA in a fashion dependent on the RNA sequence.
Transcription termination is essential for the viability of cells, and mutations in Pcf11 in yeast and in Drosophila are lethal. The disengagement of RNA polymerase II at the ends of genes is essential to reuse the polymerase and to prevent polymerase molecules from disrupting the transcriptional activity of adjacent genes. This project should provide significant insight into this fundamental cellular process. The project also provides an excellent basis for training students and postdoctoral scientists in molecular biology. The combination of biochemical and cellular approaches will provide individuals with skills needed to investigate gene regulation in living cells.