The long-term goal of this project is to understand the contribution to gene regulation made by the basic structure of the chromatin fiber, and to understand the role played by some specific and some general chromosomal proteins in generating that structure. While the bulk of the DNA is packaged in a nucleosome array, an active promoter must lie in a nucleosome-free region, a DNase I hypersensitive site (DH site). Creation of appropriate DH sites appears to be essential for gene activity. DH sites can either be "preset" before activation (as in the case of Drosophila hsp26) or be created by remodeling during gene activation (as in the case of yeast PHO5). Analysis of a large number of mutant constructs (deletions, rearrangements, and point mutations) in the hsp regulatory region indicates that the GAGA factor plays a major role in establishing the preset chromatin structure at the hsp26 promoter in Drosophila. High resolution mapping of the protein-DNA interactions of those mutant constructs that show critical alterations in chromatin structure and inducible gene expression is being carried out to establish the structural basis for these changes. The mechanism by which GAGA factor directs DH site formation will be analyzed by in vitro studies of its interactions on the hsp26 DNA with core histones, histone H1, and the TATA box-dependent transcription complex. GAGA factor activity in chromatin assembly will also be tested in vitro using a Drosophila embryo nucleosome assembly system, controlling for the above components. Mutant constructs of the hsp26 promoter (many already characterized in vivo) will be used to check the in vitro system and to confirm inferred relationships. Work is being initiated to identify a remodeling gene suitable for study in Drosophila; once such a gene is identified, a similar analysis of the determinants of its chromatin structure will be carried out. In addition to specific DNA binding proteins such as the GAGA factor, other general nonhistone chromosomal proteins may be required. We will investigate one of these, and HMG1-like protein, HMG-D. This protein will be tested to establish its association with preset and/or remodeling genes by a) examining its distribution in polytene chromosomes; b) determining whether or not it is a modifier of position effect variegation and c) analyzing its binding to DNA and nucleosomes, and its participation in nucleosome assembly and/or DH site formation in the Drosophila in vitro assembly system. Efficiency of transcription from the assembled chromatin will be assessed. Further investigations of the GAGA factor and HMG-D protein will be carried out as time and resources permit, using genetic and biochemical approaches. These studies will help to elucidate the interdependence of transcription factors and structural components of chromatin in controlling gene expression. The results should lead to a more complete model of gene regulation in eukaryotes, a critical process in normal growth and development. An understanding of these processes is essential for developing rational approaches to maintaining health and treating disease.