We are interested in understanding mechanisms of transcriptional control in eukaryotes and are currently focusing on the function and mechanism of promoter proximal pausing. Much research over the past 20 years has focused on transcriptional regulatory mechanisms that control the association of RNA polymerase II (Pol II) with a gene’s promoter. During this time, however, a small number of genes including the heat shock genes of Drosophila were found to have Pol II concentrated in the promoter region even though the genes were weakly transcribed. The Pol II at these genes has initiated transcription and paused in the promoter proximal region approximately 30 nucleotides downstream from the transcription start site. The behavior of Pol II in the promoter proximal region appears to be governed by the opposing actions of two negative regulators of elongation called NELF and DSIF and a positive regulator called P-TEFb.
Recent genome-wide mapping techniques now show that Pol II is concentrated in the promoter proximal region of thousands of genes in Drosophila and mammalian cells. Many genes involved in development, cell division, and stress responses have paused Pol II. Included among these genes are the myc and fos proto-oncogenes, and the p53 tumor suppressor gene. In addition, promoter proximal pausing represses transcription of the HIV provirus. Thus the biological and medical implications of promoter proximal pausing have dramatically increased during the past few years.
Using primarily Drosophila as a model system, we are combining biochemical, molecular genetic, and cytological methods to achieve a uniquely comprehensive approach to the study of promoter proximal pausing. We have reconstituted promoter proximal pausing in Drosophila nuclear extracts and are using this to investigate the mechanism by which specific proteins control promoter proximal pausing. Recently, we have determined that GAGA factor, a sequence specific DNA binding protein, promotes pausing by recruiting NELF to the promoter. We envision that kinetic competition between elongation and the rate at which inhibitors of elongation are able to capture the Pol II dictate the efficiency and the location of promoter proximal pausing. Support for this model has been obtained by analyzing flies that have a mutation in Pol II that slows the rate of elongation. We have discovered that the mutation shifts the location of the paused Pol II closer to the transcription start site. We often use a technique called permanganate genomic footprinting to monitor the behavior of transcriptionally engage Pol II at high resolution in living cells or tissues. To gain insight into the role of specific proteins in promoter proximal pausing, we monitor changes in the behavior of Pol II in living cells following depletion of specific proteins by RNA interference. Most recently, we have combined an in vivo crosslinking technique known as chromatin immunoprecipitation with permanganate genomic footprinting to detect paused Pol II throughout the Drosophila genome. This sets the stage for evaluating global changes in promoter proximal pausing caused by manipulating specific proteins or growth conditions in vivo.