Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci

Abstract

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.

FIG. 1

Detection of mouse Brca1 isoforms. (A) Immunoblot analysis demonstrating that mAb1, mAb2, and mAb4 recognize murine Brca1. Ten micrograms of either empty vector (pBKCMV) or pBKCMVBrca1 (mAb1) and pcDNA3.1 or pcDNA3.1-mBrca1 (mAb2 and mAb4) was introduced into 293T cells by calcium phosphate transfection. (−), empty vector; +, vector containing murine Brca1 cDNA. Cell extracts were prepared 48 h following transfection, and 50 μg of lysate was used for immunoblotting. Affinity-purified antibodies were employed at 1 μg/ml. (B) Northern analysis demonstrating the absence of full-length Brca1 transcripts in Brca1^Δ11/Δ11 MEFs. Ten micrograms of poly(A) mRNA was loaded per lane. Probes encompassing exon 11-specific sequences (right panel; see Materials and Methods) and C-terminal sequences (left panel; see Materials and Methods) were derived by PCR amplification using the mouse Brca1 cDNA as a template. (C) Immunoblot analysis of p210^Brca1 expression in wild-type and Brca1^Δ11/Δ11 MEFs. Fifty micrograms of cell extract per lane was probed with affinity-purified mAb1 at 1 μg/ml. (D) mAb1 recognizes a predominant gene product of ∼92 kDa in Brca1^Δ11/Δ11 MEFs and 210 kDa in HC11 cells. Thirty micrograms of extract was loaded per lane. (E) p92^Brca1 is expressed in testis and brain of wild-type mice. One hundred fifty micrograms of lysate per sample was subjected to SDS-PAGE on an 8% acrylamide gel. Δ11/+Brain, tissue derived from a mouse heterozygous for the wild-type and exon 11-deleted alleles of Brca1.

FIG. 2

Mouse p210^Brca1 and p92^Brca1 are cell cycle regulated. (A) Immunoblot analysis of cell cycle regulation of p210^Brca1, cyclin A, and Rad51 in serum-starved HC11 cells. Active, exponentially growing cells. (B) Immunoblot analysis of cell cycle regulation of p92^Brca1, cyclin A, and Rad51 in serum-starved MEFs homozygous for the targeted deletion of exon 11. Cells were starved as described in Materials and Methods. Cells stimulated to reenter the cell cycle by refeeding were harvested at the time points indicated. Cell extracts were prepared as described in Materials and Methods, and 10 μg of lysate was loaded per lane. Antibodies mAb1 and mAb2 revealed identical results in HC11 cells, whereas only mAb1 recognized a cell cycle-regulated band in Brca1^Δ11/Δ11 MEFs (data not shown).

FIG. 3

p210^Brca1 but not p92^Brca1 undergoes a shift in response to DNA damage. (A) Immunoblot analysis of p210^Brca1 and p92^Brca1 in cells treated with UV, gamma irradiation, or HU. p210^Brca1 exhibits a dose-dependent shift in response to UV and gamma irradiation. HC11 cells (top panel) or MEFs that express only p92^Brca1 (bottom panel) were subjected to identical treatments with UV, gamma irradiation, or HU. Twenty micrograms of lysate was loaded per lane and immunoblotted with antibody mAb1. (B) Analysis of p210^Brca1 phosphorylation in [³²P]orthophosphate-labeled HC11 cells treated with gamma irradiation (upper panel). Immediately following irradiation, HC11 cells were incubated with 5 mCi of [³²P]orthophosphate for 1 h. Three milligrams of cell extract was used for immunoprecipitation with 10 μl of the immunoglobulin G fraction of mAB1 antibody. The resolution of this assay was not sufficient to detect a mobility shift of phosphorylated products. Brca1^Δ11/Δ11 MEFs irradiated with 20 Gy received identical treatment (lower panel). (C) Immunoblot analysis of p210^Brca1 and p92^Brca1 in HC11 cells treated with 200 J m⁻² UV. p210^Brca1 and not p92^Brca1 exhibits a dose-dependent shift.

FIG. 4

Localization of p210^Brca1 and p92^Brca1 to nuclear foci. (A) Western analysis of biochemical fractionation of Brca1^Δ11/Δ11 MEFs. Equal volumes of nuclear and cytoplasmic extract were loaded per lane. Antibodies were used as described in Materials and Methods. (B) Schematic of murine Brca1 cDNA indicating regions against which antisera were raised. Numbers above the lines represent amino acid coordinates. (C) Immunofluorescence analysis of Brca1 subcellular localization. HC11 cells, wild-type MEFs, and Brca1^Δ11/Δ11 MEFs were grown on microscope slides as described in Materials and Methods. Following permeabilization, S phase cells were incubated with affinity-purified Brca1 antibodies at a concentration of 1 μg/ml.

FIG. 5

Rad51 association with p92^Brca1 is not detected in Brca1^Δ11/Δ11 MEFs. (A) Extracts generated from cycling HC11 and Brca1^Δ11/Δ11 MEFs were prepared as described in Materials and Methods. One milligram of extract was used per sample for immunoprecipitation with 2 μg of antibody. mAb1 and mAb3 were affinity purified. Rad51 antibody Ab-1 was used at 1:1,000 for Western analysis. The cross-reacting faint band observed with mAb3 in Brca1^Δ11/Δ11 MEFs does not comigrate with Rad51. (B) Seven milligrams of extract was used to detect association of p210^Brca1 with Rad51. One quarter of the extract immunoprecipitated with Rad51 Ab-1 is represented in lane 1. Immunoprecipitation of p92^Brca1 from Brca1^Δ11/Δ11 MEFs with affinity purified mAb4 does not reveal detectable Rad51 protein.

FIG. 6

Impaired Rad51 foci formation in Brca1^Δ11/Δ11 MEFs. (A) Representative Rad51 immunostained nuclei from wild-type and Brca1^Δ11/Δ11 MEFs 3 h following irradiation with 10 Gy. Cells were prepared for immunofluorescence using Rad51 antibody Ab-1 as described in Materials and Methods. Foci counts were obtained by visual inspection of 50 nuclei. (B) Graph depicting numbers of foci per nucleus following irradiation with 10 Gy at 1 h (P value = 9.1 × 10⁻¹⁷), 3 h (P value = 8.3 × 10⁻⁵¹), and 6 h (P value = 1.5 × 10⁻²⁵). (C) Rad51 levels do not change in response to irradiation in wild-type and Brca1^Δ11/Δ11 MEFs. At the time points indicated following irradiation with 10 Gy, extracts were prepared and analyzed by immunoblotting as described in Materials and Methods.

FIG. 7

Brca1 Foci are present in irradiated Brca1^Δ11/Δ11 MEFs. (A) Representative nuclei immunostained with mAb1. Cells were prepared for immunofluorescence as described in Materials and Methods. Foci counts were obtained by visual inspection of 10 to 15 nuclei. (B) Graph depicting numbers of foci per nucleus following irradiation with 10 Gy at 1 h (P value = 0.54), 3 h (P value = 0.55), and 6 h (P value = 0.24).