Genetic modifiers regulating DNA replication and double-strand break repair are associated with differences in mammary tumors in mouse models of Li-Fraumeni syndrome

Abstract

Breast cancer is the most common tumor among women with inherited variants in the TP53 tumor suppressor, but onset varies widely suggesting interactions with genetic or environmental factors. Rodent models haploinsufficent for Trp53 also develop a wide variety of malignancies associated with Li-Fraumeni syndrome, but BALB/c mice are uniquely susceptible to mammary tumors and is genetically linked to the Suprmam1 locus on chromosome 7. To define mechanisms that interact with deficiencies in p53 to alter susceptibility to mammary tumors, we fine mapped the Suprmam1 locus in females from an N2 backcross of BALB/cMed and C57BL/6J mice. A major modifier was localized within a 10 cM interval on chromosome 7. The effect of the locus on DNA damage responses was examined in the parental strains and mice that are congenic for C57BL/6J alleles on the BALB/cMed background (SM1-Trp53^+/-). The mammary epithelium of C57BL/6J-Trp53^+/- females exhibited little radiation-induced apoptosis compared to BALB/cMed-Trp53^+/- and SM1-Trp53^+/- females indicating that the Suprmam1^B6/B6 alleles could not rescue repair of radiation-induced DNA double-strand breaks mostly relying on non-homologous end joining. In contrast, the Suprmam1^B6/B6 alleles in SM1-Trp53^+/- mice were sufficient to confer the C57BL/6J-Trp53^+/- phenotypes in homology-directed repair and replication fork progression. The Suprmam1^B6/B6 alleles in SM1-Trp53^+/- mice appear to act in trans to regulate a panel of DNA repair and replication genes which lie outside the locus.

Figure 1:. Linkage analysis of mammary tumor incidence in BALB/cMed mice.

a) Linkage analysis of markers in the Suprmam1 interval associated with mammary tumors. The line indicates linkage using interval mapping relative to positions of markers in cM based on recombination maps in female mice. b) Markers used in the linkage analysis using regression model and imputed marker position of multiple qtl model along with LOD scores. The multiple QTL model was used to assess the combined linkage estimates for the Suprmam1 locus on chromosome 7 and Suprmam2 locus on chromosome 2 together.

Figure 2:. Haplotype blocks differing between BALB/cMed and strains that do not develop mammary tumors.

a) BALB/cJ haplotype blocks compared with 129S1SvimJ, FVB/NJ, C57BL/6J and DBA/2J. BALB/c is assigned a single color () and subsequent strains are assigned the same color where their haplotypes match the first strain i.e. 129S1SvimJ alleles that differed from BALB/c (), FVB/NJ alleles different from BALB/cJ and FVB/NJ (), C57BL/6 that different from 3 previous tracks () and DBA2/J alleles that is different from all 4 track above (). Region A (125-127 Mb) defines a block that is within the shoulder region of linkage bounded by polymorphisms in Plekha7 and Abca14 in Fig. 1a. Regions B (132-133.2 Mb) and C (134.5-136 Mb) lie within the peak linkage and flank Rabep2. b) Map of human chromosomes syntenic with mouse chromosome 7. The Synteny Browser was used to define relationships between mouse and human chromosomes [54]. The outer ring represents mouse chromsomes, the inner ring represents human chromsomes and the connections show syntenic regions to mouse chromosome 7. Regions of synteny to Suprmam1 interval shown with red circle in the human chromosome 10 and 16. The color key represents human chromsomes with the non-syntenic chromosomes shaded. c) Expaned view of Suprmam1 syntenic regions. The peak linkage surrounding the polymorphism in Rabep2 is syntenic with human chromosome 16. The more telomeric region of the Suprmam1 interval is syntenic with human chromosome 10. Grey bars indicate locations of genes.

Figure 3:. Generation and validation of BALB/cMed.B6-SuprMam1(N10)-Trp53^tm1Tyj/+ (SM1-Trp53^+/−) strain

a) Generation of the SM1 strain with marker assisted back-crossing. b) Mapping of SM1 congenic mouse chr7 with Affymetrix mouse diversity genotyping array. The blue circles represent BALB/cMed alleles and red circles represent C57BL/6J alleles. The SNPs shared between the strains have been removed for clarity. The region of chr7 having C57BL/6J alleles are highlighted in the box.

Figure 4:. Variation to radiation sensitivity between strains.

a) Differential expression of genes within the Suprmam1 interval in BALB/cJ and C57BL/6J mammary tissues. Expression of genes within Suprmam1 were compared for differences due to doses of (0, 4x-1.8Gy, 4x-0.75Gy) and times post-irradiation (4h, 4wks). Expression of genes within the Suprmam1 interval (upper panel) show strain-specific patterns (log2 FC >1.5 in 3 or more conditions). Selected radiation-responsive genes that are p53-dependent are included for comparison (lower panel). b) Experimental procedure for TUNEL assay in mouse mammary gland. c) Quantification of TUNEL positive cells. C57BL/6-Trp53^+/− (n = 8) mammary glands show very few TUNEL positive cells, BALB/cMed-Trp53^+/− (n = 7) and SM1-Trp53^+/− (n = 4) mammary glands exhibited high TUNEL positivity. ** p < 0.01.

Figure 5:. Single-strand annealing and gene expression differences between C57BL/6-Trp53^+/−, SM1-Trp53^+/− and BALB/c-Trp53^+/− MEFs

a) The SSA reporter construct. The construct ΔEGFP-3'-EGFP carries an acceptor EGFP gene variant, in which 46 bp encompassing the chromophore codons are replaced by an I-SecI recognition site (ΔEGFP) and a 5'-truncated EGFP donor gene (3´-EGFP). b) SSA repair efficiency in MEFs. C57BL/6J-Trp53^+/− executed significantly less repair compared to BALB/cMed-Trp^+/− whereas SM1-Trp53^+/− showed comparative repair efficiency. *** p < 0.001 . c) Heatmap showing similarity of DNA repair gene expression profiles of MEFs from three strains . p < 0.05 compares † across all strains, † BALB/cMed-Trp53^+/− and C57BL/6J-Trp53^{+/− −}, * C57BL/6J-Trp53^+/− and SM1-Trp53^+/− and * BALB/cMed-Trp53^+/− with both SM1-Trp53^+/− or C57BL/6-Trp53^+/−. d) Similarity of DNA repair gene expression pattern between SM1-Trp53^+/− and C57BL/6-Trp53^+/−. Spearman’s rank-ordered correlation analysis of qRT-PCR expression data on 24 genes from three strains show that DNA repair gene expression of SM1-Trp53^+/− is strongly correlated with C57BL/6-Trp53^+/− but not with BALB/c-Trp53^+/−.

Figure 6:. Difference in replication fork processivity between C57BL/6-Trp53^+/− , SM1-Trp53^+/− and BALB/c-Trp53^+/− MEFs

a) Replication forks of DNA synthesized by MEFs from C57BL/6J-Trp53^+/−, BALB/cMed-Trp53^+/− and SM1-Trp53^+/− during 20min pulse label with Cldu (Green) and Idu and MMC (Red). b) Representative image of the replication-fork used for measuring fork-length c) Replication-fork length is longer in C57BL/6J-Trp53^+/− MEFs compared to BALB/cMed-Trp53^+/− and comparable to SM1-Trp53^+/−. d) Replication-fork length in the presence of MMC is also longer in C57BL/6J-Trp53^+/− MEFs compared to BALB/cMed-Trp53^+/− and the fork-length is rescued in the SM1-Trp53^+/− MEFs. *** p < 0.001. Scale bar = 10μM