Activation of transcription in vitro by the BRCA1 carboxyl-terminal domain

The breast and ovarian specific tumor suppressor protein, BRCA1, has been s hown to be a transcription factor because its carboxyl terminus, when fused to the GAL4 DNA binding domain, activates gene expression in cells. In thi s study, purified GAL4-BRCA1 protein functions in transcriptional activatio n assays using a minimal in vitro system. When compared with a standard act ivator, GAL4-VP16, the levels of activation produced by the BRCA1 fusion pr otein were stronger when in the presence of certain coactivators. The trans criptional activation by BRCA1 is maximal when in the presence of the PC4 ( positive component 4) coactivator but not HMG2 (high mobility group protein 2) and when the template is negatively supercoiled. By contrast, transcrip tional activation by VP16 was highest in the presence of HMG2 as well as PC 4 and when DNA templates had linear topology. Activation by VP16 was largel y unaffected by the concentration of TFIIH, whereas activation by BRCA1 was strongly affected by TFIIH concentrations. The differing cofactor and temp late requirements suggest that GAL4-BRCA1 and GAL4-VP16 regulate different steps in the pathways that lead to transcriptional activation.