Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice

Abstract

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.

Keywords: FAN1; FANCD2; Fanconi anemia; ICL; KIN; karyomegaly.

Figure 1.

Fan1^nd/nd mice show KIN, karyomegaly, and altered ploidy. (A) Clonogenic survival analysis of MEFs exposed to MMC. For each genotype, cell viability of untreated cells is defined as 100%. Data are represented as mean ± SD. n = 3. (B) Representative images of hematoxylin and eosin (HE)-stained kidney sections from Fan1^nd/nd mice at 20 mo of age demonstrating atrophy of renal tubular epithelium, basement membrane thickening, and karyomegaly. A representative image of a kidney section stained with γ-H2AX antibodies is also shown. (C,D) The incidence of karyomegaly in 1000 cells per mouse was quantitated in three mice per genotype at 6 mo and 20 mo of age. Karyomegaly was counted when the size of a nucleus exceeded three times the size of other nuclei within the same field. Data are represented as mean ± SD. (E) DNA ploidy histograms (left panels) and separated nuclei (right panels) for kidneys from Fan1^+/nd and Fan1^nd/nd mice.

Figure 2.

Polyploidy in MEFs from Fan1^nd/nd mice upon ICL induction. Primary MEFs of the genotypes indicated were exposed to 50 ng/mL MMC for 24 h or 0.5 mM HU for 18 h before cells were fixed, stained with propidium iodide, and subjected to FACS analysis.

Figure 3.

Fan1 is not epistatic to the FA pathway with respect to ICL repair. (A,C) Clonogenic survival analysis of MEFs of the genotypes indicated after exposure to an 18-h pulse of MMC at the concentration indicated. For each genotype, cell viability of untreated cells is defined as 100%. Data are represented as mean ± SD. n = 3. (B,D) MEFs of the genotypes indicated were exposed to MMC for 18 h before cells were fixed, stained with propidium iodide, and subjected to FACS analysis.

Figure 4.

Genetic interaction between Fan1 and other ICL repair nucleases. (A,C) Clonogenic survival analysis of MEFs of the genotypes indicated after exposure to an 18-h pulse of MMC at the concentrations indicated. For each genotype, cell viability of untreated cells is defined as 100%. Data are represented as mean ± SD. n = 3. (B,D) MEFs of the genotypes indicated were exposed to MMC for 18 h before cells were fixed, stained with propidium iodide, and subjected to FACS analysis.