9808214 Kunkel Experiments in this project are designed to explore the mechanism of activation of human small nuclear RNA (snRNA) gene promoters in cultured human cells. The primary focus of this work is on the human U6 gene (although other snRNA genes will be investigated in one project). The transcription of vertebrate U6 genes is carried out by RNA polymerase III, but the promoter is constructed of a number of DNA control elements used in pol II genes. Furthermore, the U6 transcription complex is composed, in part, of factors that are shared between pol II and pol III promoters. For example, the distal region of the human U6 gene, located greater than 150 bp upstream of the transcriptional start site, contains a consensus octamer motif found in the same location as in the pol II-transcribed snRNA genes (e.g., U1 and U2 genes), and this element is bound by purified Oct-1 factor. In addition, a SPH motif that is common to many vertebrate snRNA gene enhancers is adjacent to the octamer motif in the human U6 distal promoter. The objectives of this project are: (1) the molecular characterization of human SPH-binding factor (hSBF/ZNF143), which interacts with the human U6 distal region. Using a cDNA clone encoding this transcription factor, will be investigated regions of the protein that are responsible for transcriptional stimulation and nuclear localization using transient transfection and in vitro transcription assays. (2) A comparison of the transcriptional activation by hSBF/ZNF143 of the pol III-transcribed U6 promoter and the pol II-transcribed human U2 promoter, using similar techniques. (3) The identification of protein(s) in the U6 transcription complex that interact with distal region binding factors, hSBF/ZNF143 and Oct-1. Protein-protein binding will be investigated by immunoselection using antisera specific for each protein, and the use of chemical and photoactivatable cross-linking agents. Small nuclear (snRNAs) are key components in fundamental pathways of gen e expression in eukaryotic cells. Furthermore, snRNA genes are among the most active ones in rapidly growing cells. Results from experiments in this project will provide important information regarding the mechanism of transcription of highly expressed genes. One or more objectives of this research will serve as the basis for the training of at least one graduate student and of 3-10 undergraduate students in the research laboratory.
