In eukaryotes, newly replicated DNA assembles with histones and regulatory and general transcription factors to form nucleosomes and transcription initiation complexes. Competition between the binding of regulatory factors to DNA and assembly of the same DNA into nucleosomes may play a key role in establishing and maintaining patterns of differential gene expression. The goal of this proposal is to help elucidate mechanisms which govern the outcome of this competition in vivo and guide studies of transcription using chromatin templates in vitro. Initial studies demonstrated that heat shock factor (HSF) can activate transcription while bound to a nucleosome in Saccharomyces cerevisiae. This result suggests that the position which a factor binding site adopts when it is packaged in a nucleosome influences the outcome of competitive assembly events in vivo. To test this possibility and to determine why HSF, like most regulatory factors, is usually bound to nucleosome-free DNA, we will systematically examine the influence of helical orientation and other geometric factors on the ability of regulatory factors to bind to nucleosomal DNA or, alternatively, to prevent the formation of nucleosomes. Cells will be transfected with plasmids that present cells with a choice between the binding of a regulatory factor to DNA and the assembly of the same DNA into a uniquely positioned nucleosome. HSF and the ACEI factor will be used as model regulatory factors. Results will be assessed using transcription and high resolution genomic footprinting assays. %%% Because of its great length, DNA must be extensively compacted to fit into the nuclei of cells. Compaction of DNA is accomplished by histones, proteins which associate with DNA to form nucleosomes. Nucleosomes are altered or absent from DNA in regions bound by a second class of proteins, namely those which regulate gene expression. Replication of DNA partially disrupts nucleosomes and displaces regulatory proteins. Thus, DNA must be repackaged each cell generation. Whether repackaging is by histones or by regulatory proteins may be central to whether specific patterns of gene expression are maintained or altered. However, mechanisms which govern how DNA is packaged in vivo are poorly understood. The goal of the proposed research is to elucidate these mechanisms. We will determine how the unique configuration which DNA adopts in some nucleosomes affects its packaging. The occurrence of this unique configuration, termed nucleosome positioning, suggests that if a DNA segment were forced by the binding of a rgulatory protein to adopt an alternative configuration in the nucleosome, the stability of the nucleosome would be impaired. To test whether this can account for the formation of nucleosome-free regions, we will construct a series of test DNA sequences. These sequences, when introduced into yeast, will present cells with a choice between ?a! the binding of a regulatory factor which excludes or shifts the position of nucleosomes, ?b! the assembly of a positioned nucleosome which excludes the regulatory factor, or ?c! the formation of a factor:nucleosome complex. The choice cells make will be related to the structure of nucleosomes, and interactions between regulatory proteins and DNA.
