The research plan proposed here will focus primarily on determining whether immunopurified hSWI/SNF complexes have the same subunit composition and biochemical activities as the chromatographically purified complexes. In addition, it will attempt to determine the minimal set of peptide necessary to reconstitute hSWI/SNF activity. To further characterize the hSWI/SNF complexes, cell lines that overexpress FLAG-tagged Brg1, hBrm, SNF2L, hSWI3 and Ini1 will be established using retroviral gene transfer technology. hSWI/SNF complexes will be immunopurified using an anti-FLAG affinity column, and their specific activities will be tested. To assess the nucleosomal disrupting activity of the immunopurified complexes, DNA templates will be assembled into nucleosomes, incubated with hSWl/SNF fractions and ATP, and analyzed by DNase I footprinting analysis. To study the effects of hSWI/SNF complexes on the binding activity of the TATA binding protein (TBP) and GAL4 derivatives, pre-assembled DNA templates will be treated with hSWI/SNF proteins, incubated with bacterially expressed TBP and GAL4 derivatives, and analyzed by electrophoretic mobility shift assays. In order to study the relevance of the changes caused by hSWI/SNF complexes, in vitro transcription experiments will be performed using biotinylated templates. Furthermore, to determine the subunits of the hSWI/SNF complex that are important for the nucleosomal disrupting activity, immunopurified and chromatographically purified hSWI/SNF complexes will be analyzed by SDS-PAGE and peptides that are common to all complexes will be microsequenced. Using the partial peptide sequences, degenerate oligonucleotides will be designed and used to screen human cDNA libraries for full-length SWI/SNF proteins. Finally, if time permits, experiments aimed at reconstituting the hSWI/SNF activity will be performed. hSWI/SNF cDNAs will be used to perform mixed infections of spodoptera frugipedra (Sf9) cells using various combinations, and hSWI/SNF will be immunopurified and tested for their ability to disrupt nucleosomal DNA. These studies should help clarify the role played by the SWI/SNF complex in gene regulation and will provide new insights into mechanism of action.
