Regulatory Mechanisms in Transcriptional Activation by BRAC1

摘要

Progress on determining the function of BRCA1 suggests its involvement in DNA repair and transcriptional regulation. BRCA1 C-terminus acts as a transactivation domain and introduction of germ-line mutations, but not benign polymorphisms, abolishes transcriptional activation, suggesting a critical role for this function of BRCA1 in cancer development. Our hypothesis is that BRCA1 transactivation function is regulated by an intramolecular interaction. During the past year we focused on two specific aims: (1) map the interaction sites; and (2) define the in vive dominant negative activity of truncations in BRCA1 that retain the inhibitory domain but disrupt the transactivation domain. We optimized transfection conditions and selected proper expression vectors for our analysis. In addition, we obtained several constructs containing BRCA1 fragments and confirmed protein expression in mammalian and yeast cells that will serve as important tools. A yeast two-hybrid approach and an interaction assay in mammalian cells were used to identify the interaction sites. However, while results from our interaction assay in mammalian cells are promising, the yeast two-hybrid system is not adequate due to the high background presented by the BRCA1 fragments. We have also investigated a yeast- based assay to screen for the dominant negative activity of various BRCA1 alleles.