We have been studying the Drosophila alcohol dehydrogenase (Adh) gene, focusing on the distal (adult) promoter that is regulated by the stage- and tissue-specific Adh adult enhancer (AAE). Cis-acting sequences have now ben characterized, and multiple trans-acting factors have been cloned. We have shown to single-nucleotide resolution that the distal promoter and AAE regulatory regions undergo tissue-and stage -specific sequential chromatin remodeling during development. The detailed knowledge of chromatin structure, cis-acting elements and trans-acting factors makes the Adh distal promoter a unique model system to study the molecular mechanisms of chromatin remodeling during development. We have observed an ordered nucleosomal array in early embryos disrupted or displaced in early larval fat body to expose the distal promoter and AAE as two distinct nuclease hypersensitive sites flanking a single sequence-positioned nucleosome. Distal promoter transcription in mid third instar larval fat body involves the coordinated, cooperative assembly of a multiprotein initiation complex interacting with the accessible distal promoter and AAE cis-acting sequences, juxtaposed by the positioned nucleosome. We will focus on the roles of the specifically positioned nucleosome and sequence-specific DNA binding proteins in the organization and remodeling of chromatin leading to distal promoter transcription activation. The role of the sequence- positioned nucleosome in establishing an ordered nucleosomal array covering distal promoter and AAE regulatory elements will be studied using in vitro nucleosome assembly reactions and detailed structural and compositional analyses. Interactions of specific transcription factors with their binding sites during in vitro assembly, and in preformed nucleosome templates will be investigated. Mechanisms for nucleosome structure alteration, and establishment and maintenance of nuclease hypersensitive sites will be determined; for examples, whether there are requirements for different combinations of transcription factors, and whether alterations in nucleosome core composition and posttranslational modifications of histones are involved. The formation of specific folded structure of distal transcription initiation complex juxtaposed by the sequence-positioned nucleosome will be studied. Purified components will be used to assemble the specific multiprotein complex, then parameters affecting DNA-protein and protein and protein-protein interactions, and transcription initiation will be analyzed. Finally, auxiliary factors required for chromatin remodeling will be identified and characterized by functional complementation with nuclear extracts from cell lines and embryonic stages when active developmental chromatin alterations occur.
