Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

Abstract

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.

Figure 1. Btbd12 deficiency results in growth retardation and compromised fertility

a) A representative image of Btbd12^+/+ and Btbd12^−/− littermates at 7 weeks revealing obvious growth retardation. b) Weights of male and female Btbd12^+/+, Btbd12^+/−, and Btbd12^−/− animals at 12 weeks confirming growth retardation of Btbd12^−/− mice. (n=10 for each genotype, central line represents median, box interquartile range, whiskers 90th centiles, *** p<0.001, ** p<0.01.) c) Microscopic analysis of H&E stained sections of ovary (x400, scale bar 50μm), testis (x50, scale bar 400μm) and epididymis (x400, scale bar 50μm) from Btbd12^+/+, Btbd12^−/− and Fanca^−/− animals at 16 weeks. This revealed absence of oocyte maturation in Btbd12^−/− females, and similar pattern of impaired spermatogenesis in testis of Btbd12^−/− and Fanca^−/− mice with absence of spermatozoa from epididymis.

Figure 2. Some Btbd12^−/− mice die prematurely and display brain and eye developmental defects

a) Kaplan-Meier survival curve for cohorts of Btbd12^+/+, Btbd12^+/− and Btbd12^−/− mice (n=28 per genotype, p=0.011), a sharp drop in survival is evident in the Btbd12^−/− colony occurring within the first 3 months of life. b) An increased prevalence of hydrocephalus is observed among the cohort of Btbd12^−/− mice. (**p=0.006, Fisher’s exact test; Btbd12^+/+ n=3/72, Btbd12^+/− n=13/163, Btbd12^−/− n=10/48) c) High resolution X-ray CT of representative Btbd12^−/− compared with littermate Btbd12^+/+ control, a obvious skull deformity is apparent in the Btbd12^−/− mouse (scale bar 1cm). d) H&E sectioned hydrocephalic brain from a Btbd12^−/− mouse showing dilatation of the lateral ventricles (1) and rupture of the 3^rd ventricle (3). (scale bar 1mm) e) High resolution MRI of a Btbd12^+/+ animal compared with a hydrocephalic Btbd12^−/− mouse. The image shows a large hydrocephalus (*), arrowhead points to disrupted brainstem structure (scale bar 2.5mm). f) An increased prevalence of a spectrum of eye abnormalities is observed in Btbd12^−/− mice (*** p=0.0008, Fisher’s exact test; Btbd12^+/+ n=2/71, Btbd12^+/− n=0/163, Btbd12^−/− n=11/48) g) High resolution MRI of representative Btbd12^−/− compared with wild type littermate showing unilateral anophthalmia (scale bar 2.5mm). h) Slit-lamp images revealing the spectrum of ocular abnormalities observed in the cohort of Btbd12^−/− animals (scale bar 1mm). These abnormalities range from corneal opacity (left), to microphthalmia (center) and finally anophthalmia (right).

Figure 3. A proportion of Btbd12^−/− mice develop blood cytopenias associated with genomic instability

Full blood count analysis of 4-12 weeks old Btbd12^−/− with wild type littermates a-c (n=22 for each group) a) Peripheral blood erythrocyte concentration, ns; not significant p=0.0617 b) Peripheral white cell concentration, *p=0.0155 – note a sub-population of markedly leukopenic animals. c) Peripheral thrombocyte concentration, **p=0.0027 – note a sub-population of severely thrombocytopenic animals. (in a-c central line represents mean and error bars standard error of mean) d) Proficiency of Btbd12^+/+ and Btbd12^−/− bone marrow progenitor cells to form myeloid colony forming units (CFU) from 2×10⁴ nucleated bone marrow cells. Data obtained from the average of results obtained from 3 individual mice (***p<0.0001, bar represents mean of three independent experiments and error bars standard error of mean) e) Lymphoid linage was assessed through the ability of bone marrow cells to form Pre-B cell CFU per 5×10⁴ bone marrow cells (average 3 independent mice, *p=0.017, bar represents mean of three independent experiments and error bars standard error of mean). f) Flow cytometric analysis revealing the frequency of micronucleated normochromic erythrocytes (Mn-NCE) in peripheral blood of unchallenged 16-week-old Btbd12^+/+ and Btbd12^−/− mice (n=12 per genotype, ***p<0.0001, central line represents mean and error bars standard error of mean). Micronuclei are a marker for genomic instability.

Figure 4. Btbd12 deficient cells undergo premature replicative senescence exhibiting spontaneous and inducible chromosomal instability

a) Primary MEFs obtained from Btbd12^+/+, Btbd12^+/−, and Btbd12^−/− embryos were grown in culture under normoxic conditions and monitored for population doubling. The Btbd12^−/− MEF lines prematurely cease growing. Metaphases from early passage (P3) MEFs were prepared and individually imaged. Each metaphase was then scored blind for the presence of chromosome abnormalities revealing an increased b) number of aberrations per metaphase (* p = 0.0268, t-test; bar represents mean and error bars standard error of mean) and c) an increased frequency of abnormal metaphases (**p=0.0092, Fischers’ exact test). d) Image of a single Btbd12^−/− metaphase spread revealing chromosome aberrations similar to those seen in FA cells (black arrows - radial structures; red arrows - chromatid break, scale bar 5μm). e) Cells were exposed to MMC, metaphases from these cells were scored for chromosome aberrations. MMC exposure leads to chromosomal instability in Btbd12^−/− and Fanca^−/− transformed MEFs (* p<0.05, **p<0.0001, t-test; central line represents mean and error bars standard error of mean )

Figure 5. Btbd12 deficient MEF are hypersensitive to DNA interstrand crosslinking agents

Two independent Btbd12^−/− transformed MEF lines were compared with congenic wildtype transformed MEFs for cellular sensitivity to a range of mutagens by MTS cell viability assay. Btbd12^−/− MEFs were extremely sensitivity to a) mitomycin C (MMC), b) cisplatinum, whilst they are not hypersensitive to c) UV irradiation, d) Methyl methanesulponate (MMS) or e) γ-irradiation. Btbd12^−/− MEFs were mildly sensitive to f) camptothecin (CPT). The cellular sensitivity of Btbd12^−/−, Ercc1^−/−, Fanca^−/− and Fancc^−/− MEFs were compared next to each other to g) the DNA crosslinking agent MMC and h) UV irradiation. Each point represents mean of three independent experiments, each carried out in triplicate, error bars represent standard error of the mean.

Figure 6. The interaction between Slx4 and Xpf-Ercc1 is required for crosslink repair

a) Cartoon representation of the Slx4 polypeptide, domain boundaries and interaction sites for the relevant SSEs (Xpf-Ercc1, Mus81-Eme1 and Slx1) are shown. Two deletion constructs that were predicted to disrupt the interaction with Slx1 (Slx4ΔSlx1) or the interaction with Xpf-Ercc1 (Slx4ΔErcc1) are described. b) Anti–FLAG Western blot showing the expression of FLAG-Slx4 and the truncations FLAG-Slx4ΔSlx1 and FLAG-Slx4ΔErcc1 in transfected Btbd12^−/− tMEFs. FLAG IP were then Western blotted for Ercc1, the full length and FLAG-Slx4ΔSlx1 retain interaction to this SSE subcomponent whilst FLAG-Slx4 Ercc1 abolishes this. The IP was also blotted for Mus81 showing that all truncations interact with Mus81. c) MTS cell survival of Btbd12^−/−, Btbd12^−/− + Slx4ΔErcc1 and Btbd12^−/− + Slx4ΔSlx1 strains in response to MMC. Each point represents the mean of three independent experiments carried out in triplicate and error bars represent the standard error of the mean. d) Upper Panel: Sub-cellular fractionation followed by western blot for Ercc1 in cellular fractions without DNA damage (Cyt-cytoplasmic, Nuc-nuclear, Chr-chromatin). A reduction of Ercc1 in the chromatin fraction is seen in the Btbd12^−/− cell line without DNA damage Lower Panel: Western blot analysis of Ercc1 in the chromatin fractions of Btbd12^−/− and Btbd12^+/+ tMEFs exposed to MMC (U-untreated, 4, 8). The accumulation of Ercc1 on chromatin is reduced after MMC treatment in Btbd12^−/− cells.