The cyclic AMP: CREB pathway has become a paradigm for second messenger-regulated transcriptional signaling in neurons. Phosphorylation of CREB permits binding to the co-activator CBP, which contacts proteins in the transcriptional initiation complex. Our main goal is to take advantage of a genetically-tractable system to resolve the role of CBP in transcriptional signal integration. To this end, we have cloned the Drosophila CBP homologue (dCBP) and have identified two transcription factors, dCREB-2a and cubitus interruptus (ci), that we believe utilize dCBP for gene activation. Already available mutations in the genes encoding dCBP, ci, and components of the PKA system will allow us to utilize a combination of genetic and biochemical approaches to address CBP function in the context of a living organism. We will additionally test the hypothesis that dCBP regulates gene expression not only by binding to general transcription factors, but also by recruiting proteins involved in chromatin remodeling.
Our specific aims are to: (1) Define the dCBP mutant phenotype. DCBP mutants have been generated by excising a P- element insertion located in the 5 prime untranslated region of the dCBP gene. To confirm that the lethality of these deletions is due to mutations in the dCBP gene itself, we will attempt to rescue the mutant phenotype by using a sytem that allows us to express dCBP in specific cells under the control of a yeast transactivator. We will also use this system to assess the functions of the various dCBP protein interaction domains. (2) Test whether the PKA pathway in Drosophila activates transcription through dCBP. We will test whether phosphorylated dCREB-2a interacts with dCBP by using in vitro and in vivo binding assays and determine whether dCBP activity is a component of the PKA pathway in Drosophila imaginal disc development. (3) Test whether the transcription factor cubitus interruptus (ci) utilizes dCBP for transcriptional activation. DCBP augments ci-mediated transcription in cell culture and a dCBP mutation partially suppresses a dominant ci mutation in intact flies. We will determine whether dCBP mutations alter the phenotypes of hypomorphic and dominant ci gain-of-function mutations. We will also determine whether dCBP controls the expression patterns of wingless and decapentaplegic, which are believed to be downstream from ci. (4) Determine the role of 'chromatin organizers' in dCBP-mediated transcription. Recent evidence indicates that CBP interacts with a GCN5-like histone acetylase designated PAF. A Drosophila homologue of SIR2, which mediates silencing in yeast, binds to the dCBP 'CREB-binding domain'. We will test whether these putative 'chromatin organizers' are recruited to a promoter through dCBP and determine the functional consequences of these interactions.
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