Evidence for a transcriptional activation function of BRCA1 C-terminal region

Abstract

Mutations in BRCA1 account for 45% of families with high incidence of breast cancer and for 80-90% of families with both breast and ovarian cancer. BRCA1 protein includes an amino-terminal zinc finger motif as well as an excess of negatively charged amino acids near the C terminus. In addition, BRCA1 contains two nuclear localization signals and localizes to the nucleus of normal cells. While these features suggest a role in transcriptional regulation, no function has been assigned to BRCA1. Here, we show that the C-terminal region, comprising exons 16-24 (aa 1560-1863) of BRCA1 fused to GAL4 DNA binding domain can activate transcription both in yeast and mammalian cells. Furthermore, we define the region comprising exons 21-24 (aa 1760-1863) as the minimal transactivation domain. Any one of four germ-line mutations in the C-terminal region found in patients with breast or ovarian cancer (Ala-1708-->Glu, Gln-1756 C+, Met-1775-->Arg, Tyr-1853 ->Stop), had markedly impaired transcription activity. Together these data underscore the notion that one of the functions of BRCA1 may be the regulation of transcription.

Figure 1

Structure of fusion proteins. Fragments 16/24, 16/23, 16/22, 16/21, 21/24, 22/24, and 21/22 contain exons 16–24 (aa 1560–1863, top bar), 16–23, 16–22, 16–21, 21–24, 22–24, and 21–22, respectively, of the BRCA1 gene fused to the DNA binding domain of GAL4 (black box). Fragments 16/24 (Gln-1756 C+) and 16/24 (Tyr-1853 → Stop) carry insertions reported to truncate BRCA1. Fragment 16/24 (Ala-1708 → Glu) is a substitution reported in patients. Fragments 16/24 (Met-1775 → Lys, Met-1775 → Thr, Met-1775 → Arg, Met-1775 → Glu, and Met-1775 → Val) are each individual mutations at codon 1775, with the Met-1775 → Arg mutant reported in patients with breast cancer. TA 16/24 represents exons 16–24 fused to the activation domain of GAL4. Arrowheads indicate the positions of the mutations, and shaded areas represent truncated parts of the mutant protein.

Figure 2

Transcriptional activation by BRCA1 C terminus. Growth curves of S. cerevisiae (HF7c) carrying the following fragments of BRCA1 in pGBT9 grown in medium lacking tryptophan and histidine: 16/24 (□), 16/23 (▪), 16/22 (○), 16/21 (•), 21/24 (▵), 22/24 (▴), and 21/24 (×). (Inset) Growth in medium lacking tryptophan only. OD₆₀₀ of the cultures (y axis) is plotted against time in culture (x axis).

Figure 3

Mutations occurring in patients impair activity. Shown are growth curves of S. cerevisiae (HF7c) carrying the following fragments and mutants in pGBT9 grown in medium lacking tryptophan and histidine: 16/24 (□), 16/24 (Ala-1708 → Glu) (▴), 16/24 (Gln-1756 C+) (○), 16/24 (Met-1775 → Arg) (•), and 16/24 (Tyr-1853 → Stop) (×). (Inset) Growth in medium lacking tryptophan only. The OD₆₀₀ of the cultures (y axis) is plotted against time in culture (x axis).

Figure 4

Transcription activation by BRCA1 C terminus in mammalian cells. Luciferase activity of the following fragments and mutants in pSG424, transfected along with luciferase reporter plasmid into Cos-7 cells plotted as fold activation over control: vector, pSG424 alone; 16/24, wild-type exons 16/24; 16/24 (Met-1775 → Arg), Met-1775 → Arg mutation; 16/24 (Gln-1756 C+), cytosine insertion at position 1756. Cells were harvested at 24 hr (black bars) and 48 hr (shaded bars).