Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation

Abstract

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus. Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.

Figure 1. Expression levels of loss-of-function BRCA1 mutants

Cell lysates containing comparable cell numbers were separated on SDS-PAGE. At least two independent transformants were assayed for each mutant to control for clonal variation. LexA fusion mutant proteins expressed in yeast were detected by western blot using a monoclonal α-LexA antibody. A schematic representation of the fusion proteins bearing missense mutations (white arrow; M.W. 65 kDa) and a nonsense mutation (gray arrow; M.W. 49 kDa) is shown. Note that mutation Y1769X disrupts the BRCT-C but retains BRCT-N.

Figure 2. Deletion analysis of the C-terminal region

A. Alignement of the wild-type sequences of the C-terminus of rat, mouse, dog and human BRCA1. Amino acids in bold represent conserved residues. The shaded area corresponds to residues at the 3’ border of the BRCT-C domain. B. Transcriptional activity of GAL4 DBD fusion deletion constructs, made in the context of BRCA1 aa 1560–1863. S. cerevisiae (HF7c) carrying the indicated fusion proteins were assayed for growth in the absence of tryptophan and histidine in liquid medium. Activity relative to cells growing in medium lacking tryptophan alone after 36 hours is shown in parenthesis. Filter β-galactosidase assays for SFY526 were scored at 12 hours after X-gal addition. At least 4 independent clones were assayed for each construct. C. Western blot showing levels of protein expression of the different constructs (black arrow) detected by a α-GAL4-DBD monoclonal antibody.

Figure 3. Domain structure of BRCA1 C-terminal region (aa 1560–1863) and characterized mutations

Top panel shows a schematic representation of full length BRCA1 protein featuring the RING domain (yellow box) in the N-terminal region and the BRCT domains (red circles) in the C-terminal region. The region analyzed in this study is contained in the red box which is enlarged and represented in the bottom panel. Purple and pink bars represent predicted β-strands and α-helices, respectively. Secondary structure predictions were made by Zhang et al. (36) based on the crystal structure of XRCC1 BRCT domain. Mutations represented on the upper part (red triangles) result in loss of function whereas mutations on the lower part (green triangles) result in activity equal or higher than wild-type. Germ-line mutations and polymorphisms are variants defined by genetic linkage to be disease-associated and benign polymorphisms, respectively. Site-directed mutagenesis, PCR mutagenesis, transposon-mediated mutagenesis and deletion analysis represent mutations that have been characterized by transcription activation assay in yeast to be either loss of function (upper part) or wild-type (lower part).