Establishment and maintenance of appropriate transcriptional regulation is essential to proper development and differentiation, and mistakes in establishing appropriate transcription patterns are linked to disease states such as cancer. Promoter regions of inactive genes in eukaryotes are assembled into nucleosomes (and frequently higher order structures); the presence of nucleosomes inhibits many steps in the transcription process. Extensive genetic, structural and biochemical evidence suggests that the dynamic remodeling of nucleosome structure over a promoter during transcriptional activation is likely to be a key component of gene regulation. Protein complexes have been characterized in eukaryotes that remodel nucleosome structure, and biochemical and genetic data suggest that they might be a necessary component of transcriptional activation in chromatin. These complexes all contain a protein related to the yeast SW12/SNF2 protein. The SW12/SNF2 protein is a member of a multiprotein complex which is capable of altering nucleosome structure and facilitating transcription factor binding to nucleosomes, both in an ATP-dependent manner. Similar ATP-dependent nucleosome remodeling activities have been found with the human SWI/SNF (hSWI/SNF) complexes and with a Drosophila activity termed NURF that includes a SW12/SNF2 related gene called ISWI. The investigator proposes to characterize the SW12/SNF2-related complexes in human cells, and to test the hypothesis that these complexes might play key roles in transcriptional activation. There are three genes that have been identified to date in humans with high degrees of similarity to yeast SWI2/SNF2: BRG1 and hBRM have extensive similarity throughout the protein, and hSNF2L has similarity in the ATPase domain and has extensive similarity throughout with the Drosophila gene ISWI. He proposes to continue the purification of complexes that contain these proteins, and to characterize whether these complexes associate with RNA polymerase II. He will compare the ability of these complexes to alter nucleosome structure, to facilitate transcription factor loading, and to modulate transcription initiation and elongation on nucleosomal templates. He will express dominant negative versions of these proteins in a conditional manner in mammalian cells to determine whether these mutants alter differentiation of muscle cells or expression of the heat shock loci. The experiments described in this proposal will provide a biochemical characterization of SWI/SNF related complexes in humans, and will identify natural targets of those complexes.
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