Brca1 in immunoglobulin gene conversion and somatic hypermutation

Abstract

Defects in Brca1 confer susceptibility to breast cancer and genomic instability indicative of aberrant repair of DNA breaks. Brca1 was previously implicated in the homologous recombination pathway via effects on the assembly of recombinase Rad51. Activation-induced cytidine deaminase (AID) deaminates C to U in B lymphocyte immunoglobulin (Ig) DNA to initiate programmed DNA breaks. Subsequent uracil-glycosylase mediated U removal, and perhaps further processing, leads to four known classes of mutation: Ig class switch recombination that results in a region-specific genomic deletion, Ig somatic hypermutation that introduces point mutations in Ig V-regions, Ig gene conversion in vertebrates that possess Ig pseudo-V genes, and translocations common to B cell lymphomas. We tested the involvement of Brca1 in AID-dependent Ig diversification in chicken DT40 cells. The DT40 cell line diversifies IgVlambda mainly by gene conversion, and less so by point mutation. Brca1-deficiency caused a shift in Vlambda diversification, significantly reducing the proportion of gene conversions relative to point mutations. Thus, Brca1 regulates AID-dependent DNA lesion repair. Interestingly, while Brca1 is required to recruit ubiquitinated FancD2 to DNA damage, the phenotype of Brca1-deficient DT40 differs from the one of FancD2-deficient DT40, in which both gene conversion and non-templated mutations are impaired.

Fig. 1

Brca1-deficient derivatives of DT40 cre. (A) Southern blot. (B) Targeting strategy. (C) Western blot of nuclear lysates, blotted and probed with anti-serum to chicken Brca1. The position of Brca1 is indicated. Other bands are non-specific proteins recognized by either the primary antiserum or secondary anti-rabbit IgG used for detection. (D) Flow cytometry of cells stained for surface IgM a – DT40 cre b – DT40 cre Brca1+/- c – DT40 cre Brca1-/- clone #100 d – DT40 cre Brca1-/- clone #125. One to two-week old cultures of each genotype were stained with anti-chicken IgM labeled with FITC, and analyzed by flow cytometry. All cultures are comprised of largely IgM+ cells.

Fig. 2

Frequency of IgM loss and patterns of non-templated point mutations in Vλ in DT40 cre and derivatives deficient for Brca1. (A) Frequency of IgM loss. Each symbol represents the percentage of IgM-loss variants in an individual subclone after two (filled symbols) or four (open symbols) weeks of culture. The median percentage of IgM-loss of all subclones of a particular genotype is plotted as a large dash, with the value indicated above each set. (B) Patterns in Vλ of non-templated point mutations in cells of DT40 cre or Brca1- /- derivatives either selected, or not, for lack of surface IgM. Numbers are raw numbers of each class of base change.

Fig. 2

Frequency of IgM loss and patterns of non-templated point mutations in Vλ in DT40 cre and derivatives deficient for Brca1. (A) Frequency of IgM loss. Each symbol represents the percentage of IgM-loss variants in an individual subclone after two (filled symbols) or four (open symbols) weeks of culture. The median percentage of IgM-loss of all subclones of a particular genotype is plotted as a large dash, with the value indicated above each set. (B) Patterns in Vλ of non-templated point mutations in cells of DT40 cre or Brca1- /- derivatives either selected, or not, for lack of surface IgM. Numbers are raw numbers of each class of base change.

Fig. 3

Mutation distributions in randomly selected DT40 cre and Brca1-deficient derivatives. Each horizontal line is a map of events in one individual clone plotted with respect to the location in a 427-basepair region of Vλ. Nucleotide positions are indicated on the X-axis, and the three gray rectangles indicate the location of the three CDRs. Mutations from cells sorted for IgM loss are shown in plots (A) and (B), and from unsorted populations in plots (C) and (D). Filled circles represent non-templated point mutations, black lines represent gene conversions, filled trapezoids and triangles represent insertions and deletions, and gray squares are ambiguous events.

Fig. 4

Frequency of IgM loss and mutation pattern in DT40 and derivatives deficient for XRCC3 or Brca1. (A) Frequency: Two independent experiments are shown. Each symbol represents the percentage of IgM-loss variants in an individual subclone after five weeks of culture. The median percentage of IgM-loss of all subclones of a particular genotype is plotted as a large dash, with the value indicated above each set. (B) Patterns of non-templated point mutations in Vλ, with or without selection for surface IgM phenotype. Raw numbers (% of total mutations).

Fig. 4

Frequency of IgM loss and mutation pattern in DT40 and derivatives deficient for XRCC3 or Brca1. (A) Frequency: Two independent experiments are shown. Each symbol represents the percentage of IgM-loss variants in an individual subclone after five weeks of culture. The median percentage of IgM-loss of all subclones of a particular genotype is plotted as a large dash, with the value indicated above each set. (B) Patterns of non-templated point mutations in Vλ, with or without selection for surface IgM phenotype. Raw numbers (% of total mutations).

Figure 5

RT-PCR analysis of Rad6 and PCNA in DT40 cells deficient for Brca1. As compared with the control gene, β-actin, no difference in expression of Rad6 and PCNA was observed in DT40 Brca1-/- cells. AID-/- cells are in the DT40 cre background and were used as an additional control.