Functional Analysis of Breast Cancer Susceptibility Genes in Mice

Division Of Basic Sciences - Nci

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Abstract

Functional studies of BRCA2 variants using our mouse ES cell-based assay have proven to be very valuable for the functional classification of variants of unknown clinical significance. Such assays provide clues to the impact of these variants on cell viability, repair of DSBs by HR or sensitivity to DNA damaging agents. However, predisposition to cancer due to inheritance of these variants cannot be determined by these in vitro assays. The physiological impact of pathogenic variants and disease risk can be best studied in an in vivo model system. Over the years, we and others have generated several Brca2 mouse models to examine the impact of BRCA2 variants or loss of a functional domain. One of the variants, Phe2406Asp, was shown by in vitro binding studies to disrupt the binding of BRCA2 with meiosis specific recombinase, DMC1. It was shown that altering phenylalanine to any other amino acid disrupted the interaction between the two proteins. We generated a knock-in mouse model where we substituted the hydrophobic phenylalanine at position 2351 of mouse BRCA2 (corresponding to human 2406 residue) with a hydrophilic and charged aspartic acid residue. Surprisingly, the mutant mice were found to be fertile, and the spermatocytes showed normal DMC1 foci suggesting that this residue was not as critical for DMC1 recruitment as suggested by the in vitro studies. In another study we had identified a novel BRCA2 transcript lacking exons 4-7, which we showed was proficient in DNA repair, but it was unclear if it is fully functional like WT BRCA2. To address this, we have generated mice lacking Brca2 exons 4-7 and found mutant mice to be indistinguishable from wild-type littermates. We have also generated a number of mouse model with single amino acid substitutions. We have generated mice with a glycine to arginine substitution (Gly25Arg) in the PALB2 (Partner and localizer of BRCA2) binding domain of BRCA2 and showed the importance BRCA2-PALB2 interaction in the repair of DSBs by HR as well as protection of stalled replication forks. We have also examined the physiological significance of BRCA2-DSS1 (Deleted in split hand/split foot1) interaction using a knock-in mouse model with a leucine to proline change in codon 2431 (Leu2431Pro, equivalent to human Leu2510Pro), which weakens this interaction. Our functional studies in mESC showed that this interaction is critical for RAD51 recruitment and repair of DSBs by HR. Surprisingly, our functional studies in mice revealed that the interaction is dispensable for repair of DNA replication induced breaks as well as repair of DSBs during meiosis. When bound to BRCA2, DSS1 has been reported to act as a DNA mimic and facilitate RAD51 recruitment by displacing Replication Protein A (RPA) from single-stranded (ss) DNA at DSBs. Our findings suggest that the presence of homologous chromosomes in proximity to DSBs during early prophase I stage of meiosis may compensate for the defect in BRCA2-DSS1 interaction. This is supported by our observation that tethering a biotinylated homologous DNA to the site of DSBs using streptavidin-fused Cas9 partially rescued the HR defect in mESC expressing this variant. Homology-based structural modeling and our functional studies in mESC revealed one of the variants, Arg3052Gln, to have a moderate effect on the structure and DNA repair function of BRCA2. This residue is located in the C-terminal DNA binding domain of BRCA2. Notably, this variant was predicted to be a neutral variant in a large-scale variant classification study utilizing personal and family history data and co-segregation with disease data. We are currently characterizing a knock-in mouse model expressing Arg2971Gln (equivalent to human Arg3052Gln) to determine whether the moderate defect in BRCA2 contributes to cancer susceptibility. We are also generating mouse models to functionally characterize another important domain of BRCA2, the BRC domain containing eight BRC repeats spanning over 1100 amino acids. Several of these repeats have been shown to bind to RAD51. Each repeat consists of 35-40 amino acids with an evolutionarily conserved core consisting of hydrophobic residues that facilitate close contact with RAD51. In spite of such an essential function, no cancer causing missense substitution has been identified in this region, which suggests functional redundancy between these repeats. We and others have shown that a single BRC is sufficient for RAD51 recruitment and has partial HR activity in mESC. We are generating mice with a single BRC, repeat 2 or repeat 4, to examine whether the residual HR activity is sufficient for normal physiological functions.