The SAGA complex is a highly conserved transcriptional co-activator comprised of over 20 protein subunits. SAGA contains 2 enzymatic activities, a histone acetyltransferase and a histone deubiquitinase, interacts with sequence-specific DNA binding transcription factors, and facilitates the formation of preinitiation transcription complexes. SAGA is linked to the functions of several oncogene and tumor suppressor gene products, and disruption and/or misexpression of SAGA subunits is observed in many forms of cancer and can give rise to neurodegenerative disease. While SAGA was discovered and most widely studied in yeast, it is not essential in that organism. By contrast, SAGA is essential for the development of multicellular eukaryotes, yet very little is known about its functions in developmental programs of gene expression. This application proposes to combine genetic and tissue-specific tools in the model organism, Drosophila melanogaster, with powerful proteomic and genomic approaches to dissect the functions of SAGA in cell-type and tissue-specific gene expression. The first specific aim will complete the functional and phenotypical analysis of recently identified but as yet uncharacterized Drosophila SAGA subunits. These include the fly homolog of the human ATXN7 protein, which when perturbed gives rise to spinocerebellar ataxia 7, and two splicing proteins that may link SAGA to RNA splicing. In the second aim, we propose to express epitope tagged versions of SAGA subunits in specific tissues and determine the target genes of SAGA in each tissue using chromatin immunoprecipitation and high throughput sequencing. The dependence of expression on each of the SAGA target genes on each of the SAGA activities (acetyltransferase, deubiquitinase, coactivator) will be measured in the appropriate SAGA mutant embryos. Lastly, we have identified a number of tissue-specific transcription factors that co-purify with SAGA. These transcription factors will be examined for their roles in recruiting SAGA to their respective target genes. Thus, this project will establish a comprehensive view of SAGA localization and function in specific tissues in a developing multicellular eukaryote. We anticipate that analysis of the mechanisms by which SAGA regulates tissue-specific gene expression will establish paradigms for SAGA function in metazoans, and advance our understanding of its roles in cancer and neurodegenerative disease.
The SAGA protein complex is disrupted or misregulated in several cancers and in neurodegenerative disease. This project seeks to elucidate the normal functions of the SAGA complex in the development of multi-cellular organisms using fruit flies as a model system to gain insights into how its perturbation contributes to human disease.
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