There is much current interest in isolating nuclear proteins that bind to specific DNA sequences in and around eukaryotic genes. The rationale behind this often derives from data that show the sequence elements bound are important in mediating expression or regulation of the gene in question. However, it is increasingly clear that the question of whether the particular DNA-protein interaction under investigation actually occurs in vivo in a given tissue should be addressed. Often specific factors are unable to bind to their recognition sequences because a precise pathway of chromatin assembly has excluded them from the nucleoprotein complex. Moreover, distinct protein species may recognize identical sequence elements, but in a given cell type only one of them may be utilized for gene regulation. In this light we plan to use rapid and sensitive genomic footprinting techniques recently developed in this laboratory to study, at single base resolution, the in vivo nucleoprotein organization and specific binding patterns characteristic of Drosophila locus 67B1. In this manner it is expected that important regulatory sequences and details of their binding interaction will uncovered. Consisting of at least seven closely-linked genes that are hormonally regulated in differing tissue-and developmental-specific patterns (in addition to thermal induciblity in virtually all tissues) locus 67B1 is obviously highly complex in regulatory terms. Organization at hsp22 and hsp27 is of particular interest since their expression patterns show elements of both coordinate and independent modes of developmental regulation. With a precise developmental and tissue-specific description of the effects of hormone on the in vivo nucleoprotein organization, together with the results of DNA sequence-related effects obtained via P-element mediated transformation, it is expected that this project will provide the detailed baseline to which in vitro binding and transcription assays must be referred for relevance. We plan to initiate such in vitro work with extracts prepared from both hormonally naive and exposed cells and tissues, with a long term goal of recapitulating aspects of the hormonal regulation imposed upon these genes. Understanding what actually happens in the cell as contrasted to what can happen in a test tube is often difficult. The results of this in vivo study of a gene family with a complex regulatory pattern should give insight into some of the those potential differences and, with luck, the reasons for those differences.
