We wish to understand the chromatin structure of an inducible gene, taking as our model system the heat shock gene hsp26 in Drosophila. This gene, while normally quiescent, can be activated within a few minutes in all cell types examined in response to the shock or other stresses. Based on many well-studied examples, a general picture of the chromatin structure of active/inducible genes has emerged in recent years. Several distinctive features are seen: (i) the entire active gene, and several kilobases of surrounding DNA, show an alternative packaging, indicated by an elevated sensitivity to DNase I; this appears to reflect both an altered local nucleosome structure and/or altered higher order nucleosome packaging; (ii) the nucleosome array shows a pattern of discontinuities, sites hypersensitive to DNase I being observed 5' (and sometimes 3') to the gene; and (iii) specific NHC proteins required for transcription are associated with the promoter region 5' to the gene. The research proposed here will characterize the DNA structural elements and the proteins that bind to them that together define these characteristics of the hsp26 gene. The process of chromatin assembly will be analyzed in vitro and subsequently studied in vivo to identify regulatory features. Specifically, we intend to carry out the following studies: 1. Completion of an analysis of the protein-DNA interactions in the region 5' of hsp 26 for wild-type and mutant alleles. 2. Identification and characterization of nonhistone chromosomal proteins binding to this region of DNA. 3. Identification of DNA elements essential for formation of the DNase I hypersensitive sites (DH sites) and for effective utilization of the promoter in vivo. 4. Continued development of an in vitro transcription system based on nuclear extracts from the embryo, and development of an in vitro assembly procedure capable of generating the specific DH sites and other features of active chromatin structure observed. 5. Characterization of """"""""nucleosomal"""""""" proteins thought to be associated with some inducible and/or active genes. Initially we will focus on a putative HMG protein and on a variant of histone H2A. The sum of these experiments should allow us to describe in detail the chromatin structure of the hsp 26 gene, to analyze the mechanism of gene activation, and to begin to analyze the mechanisms that direct specific features of chromatin assembly to generate a transcriptionally competent template. These studies should lead to a better understanding of how gene expression is controlled in eukaryotes, basic formation for understanding normal and abnormal cell growth.
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