Germline mutations in BRCA1 account for 50% of familial breast cancer and 5-10% of breast cancer cases with an early age of onset. BRCA1 encodes a novel protein implicated in the cellular response to DNA damage, with possible roles in homologous recombination, as well as transcriptional regulation. As an initial step to understanding its functional properties, we have established cells with tightly regulated inducible expression of full length BRCA1, and have identified downstream targets using a microarrayed oligonucleotide screening strategy. We have observed that overexpression of BRCA1 induces expression of the DNA-damage responsive gene GADD45, which in turn activates JNK/SAPK-dependent apoptosis. In this proposal, we will expand on these BRCA1 overexpression studies and develop a loss-of-function model. First, we will use cells with inducible expression of a tagged BRCA1 construct to investigate the post-translational changes in BRCA1 that occur following DNA damage, including phosphorylation, and alterations in size of the in vivo BRCA1 complex and associated proteins. Having identified GADD45 as a BRCA-1 target gene, we will analyze the role of BRCA1 in transcriptional regulation by defining the BRCA1-responsive sequences in the GADD45 promoter, searching for DNA-binding protein(s) that mediate the induction of GADD45 by BRCA1, and characterizing the nature of their interaction with BRCA1 itself. The requirement for GADD45 to mediate the effects of BRCA1 will be tested using a previously defined antisense strategy. Second, we will develop dominant negative constructs to investigate BRCA1-dependent DNA damage response pathways and their contribution to mammary tumorigenesis. Our strategy will be to generate fusion proteins containing the protein interaction """"""""ring"""""""" domain of BRCA1 or its associated protein BARD1, fused to a heterologous domain leading to altered subnuclear localization. The effectiveness of these dominant negative constructs will be assayed by immunofluorescence and immunoprecipitation studies. Successful dominant negatives will be expressed in cell lines and in primary cells to identify DNA damage responsive genes whose expression is dependent upon BRCA1 function. To analyze the consequences of BRCA1 inactivation in developing mammary tissue, transgenic mice will be generated using MMTV-driven dominant negative constructs. By analysis of altered gene expression profiles following BRCA1 overexpression, together with the disruption of BRCA1 function using dominant negative constructs, we expect to gain insight into the function of this tumor suppressor.
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