To understand the regulation of normal cell growth and development and how failure to appropriately regulate these processes leads to abnormal or diseased states, it is necessary to determine the mechanisms by which the cellular transcription machinery recognizes gene promoters and initiates transcription. The regulation of pol II gene expression requires transcriptional activators and the polymerase- associated general transcription factors (GTFs) to associate with the DNA regulatory sequences that control gene expression. In vivo, DNA is complexed and condensed with histones and nonhistone proteins to form chromatin, the basic unit of which is the nucleosome. Numerous studies have indicated that, in general, nucleosomes and chromatin components inhibit transcription factor binding to DNA, transcription initiation, and transcription elongation. Understanding how the cell makes DNA sequences accessible for transcription factor function is an issue of central importance in gene regulation and the regulation of cell growth, development, and differentiation. Genetic and biochemical studies indicate that histone hyperacetylation and the human SWI/SNF (hSWI/SNF) comples, which alters nucleosome structure in an ATP dependent manner, can facilitate some steps in the process of gene activation in chromatin. The SWI/SNF complex appears to be conserved from yeast to humans, and constituent subunits and related homologues have been implicated in cell growth, embryonic development, differentiation, and retroviral integration, in addition to transcriptional activation. Similarly, the correlation between the presence of hyperacetylated histones and genes being actively transcribed extends to all eukaryotes. The purpose of this proposal is to characterize how alterations in chromatin structure mediated by the hSWI/SNF complex or histone hyperacetylation facilitate the function of transcriptional factors in chromatin. In vitro studies will combine purified transcriptional activators and chroatin components with in vitro reconstitution techniques to characterize binding of GTFs and activators to defined nucleosomal templates in the presence and absence of hSWI/SNF and/or hyperacetylated histones. The ability of GTFs and activators, alone and in combination, to further alter the structure/stability of the nucleosome, and to initiate transcription from the altered template will also be examined. To examine the effects of hSWI/SNF on transcription in vivo, genes encoding dominant negative versions of hSWI/SNF components will be conditionally expressed in mammalian cells to determine whether gene activation by nuclear hormone receptors is affected.
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