Transcriptional regulation is a key process controlling cell growth, determination and differentiation during development. Perturbation of transcription factor function can lead to developmental disorders such as Aniridia and Waardenburg syndrome (Pax gene mutants) and cancer (p53, Myx, etc.). In higher eukaryotes, genomic DNA is in the form of chromatin. Altered expression of proteins affecting chromatin structure and DNA-accessibility can also contribute to cancer (ALL-1/HRX, bmi-1). Molecular understanding of normal transcription factor function in vivo is required to appreciate these abnormal (disease) situations. The broad, long term goal of this project is to understand how transcription factors interact with chromatin and proteins regulating chromatin structure; e.g., whether regulated access to DNA serves to specify transcription factor activity in different tissues. The project is focused on a single, evolutionarily conserved transcription factor, CCAAT/Enhancer Binding Protein (C/EBP). Drosophila C/EBP (DmC/EBP) will be studied to take advantage of existing mutants and the advanced molecular genetics of this model system. The following hypothesis is submitted and aspects of it will be tested: DmC/EBP acts as a """"""""switch"""""""" for terminal differentiation; its tissue-specific activity (activation of target genes) is regulated by restricted access to specific binding sites in chromatin. A novel screen will be used to identify genes that, when expressed in the cells normally expressing DmC/EBP, increase or decrease the biological activity of DmC/EBP. Polycomb- (Pc) and Trithorax- (Trx) group genes, which regulate chromatin structure and inducible transcription will be tested for genetic interactions with DmC/EBP. DmC/EBP target genes will be identified by screening enhancer-trap lines for inserts affected by a DmC/EBP null mutation; and endogenous regulated genes will be cloned. Lastly, it will be determined whether the ability of ectopically expressed DmC/EBP to activate target genes is tissue-restricted, and whether any such ability is directly influenced by Pc-g and Trx-g gene products. Future work will explore the biochemical interactions between DmC/EBP and chromatin.
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