Characterization of RBT1, a novel member of the TRIP-Br family of transcriptional modulators

Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We have identified a novel RPA protein interactor, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that over-expressed green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to normal non-immortalized human mammary and bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian transactivation data are indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. NIH3T3 mouse fibroblasts stably overexpressing RBT1 meet several criteria of transformed cells, including lack of contact growth inhibition, growth of colonies in soft agar and tumor formation in nude mice; HCT116 colon carcinoma cells overexpressing RBT1 exhibit an enhanced growth rate in culture and form extremely aggressive tumors in vivo.;We propose that RBT1 is a novel transcriptional co-activator that interacts with several important cellular proteins including RPA and cdk4, and that RBT1 may promote tumorigenesis when its protein product is over-expressed. Enhancement of E2F1 transcriptional activity by overexpressing RBT1 suggests that this novel protein has implications on cell cycle regulation and progression.;BLAST analysis of the human genome localizes RBT1 to chromosome 19g13.1-q13.2 adjacent to the coding region for TRIP-Br1/p34SEI-1 in a region characterized by frequent amplification in ovarian carcinomas. Further analysis of amino acid homology places RBT1 in the TRIP-Br protein family consisting of at least four members (RBT1, TRIP-Br1 (p34SEI-1), TRIP-Br2, and HEPP (cdca4)). While not much is known about this protein family, TRIP-Br1 has been found to interact with the cyclin-dependent kinase, cdk4, and modulates E2F transcriptional activity (Hsu et al., 2001). In vitro binding and co-immunoprecipitation assays suggest that RBT1 interacts with at least cdk1, cdk2 and cdk4, and modulates E2F1 transcriptional activity indicative of one possible mechanism by which higher levels of RBT1 can both promote and enhance tumorigenesis.