Origin, location, segregation, and implications of the FAAP100 variant.

(A) Pedigree of a fetus with FA-suggestive phenotype (III-2, affected individual; solid and slashed circle, marked with a red arrow, ID no. 1176) and family. Squares denote males; circles denote females. Genotyped individuals of the core family are indicated by an asterisk, unassessed genotypes by a question mark, and proven or obligate heterozygotes by half-filled symbols. (B) Five FA (prefix FANC) and FA-associated (prefix FAAP) genes in major autozygous regions (black blocks) on 3 different chromosomes (Chr) in the affected III-2 (1176) are shown. (C) Sanger electropherograms identified a homozygous A>C variant sequence, heterozygous sequence, or WT sequence at FAAP100 position c.1624 (highlighted by a red frame), predicting p.(T542P), in relatives of the core family as indicated. (D) Cell-cycle analysis of cultured amniotic fibroblasts by flow cytometry. Exemplary individual measurements. Histograms of cells from a normal control (non-FA; top), a fetus with FA (positive control; middle), and fetus III-2 (1176; bottom). Black histograms are from untreated cultures and superimposed gray or gray/red histograms are from cultures exposed to MMC (10 ng/mL, 48 hours), shown individually in Supplemental Figure 1C. The percentage of cells in G₂ phase is indicated. Red coloring and arrows denote an increased G₂ compartment size. Variability and significance of G₂ phase arrest in repeated measurements are shown in Supplemental Figure 4A. (E) Titration experiments show reduced levels of FAAP100 protein in FAAP100^T542P-mutant 1176 cells from affected III-2 cells (approximately 3 steps of 1:3 dilution each lower than in non-FA cells). Reduced levels of FANCL are also suggested in 1176 cells (approximately 2 steps of 1:3 dilution lower than in non-FA cells). Wedges represent dilutions at the ratios indicated above the blots. FA-A, cells from an individual with FA, subtype A. Vinculin was used as a loading control.

Ectopic expression of FAAP100^WT or FAAP100^T542P in FAAP100-deficient cells.

(A) Metaphase micrographs of FAAP100^T542P-mutant 1176 cells after exposure of the cultures to MMC (100 ng/mL, 48 hours). Parental 1176 cells and mock- or mutation-transduced (+vector^pLVX or +FAAP100^T542P) 1176 cells show distinctly increased chromosome breakage, mostly of the chromatid type, whereas WT transduced 1176+FAAP100^WT cells are rescued. Radials are marked with red arrows. (B) Box plots reflect the proportion of cells with the indicated number of chromosome breaks per metaphase; single value (♦), median (─), mean (□), IQR (─), minimum (×), and maximum (×) for the number of breaks from 3 independent experiments; blue symbols are from untreated cultures, and red are from cultures exposed to MMC (100 ng/mL, 48 hours). Light gray shading indicates high rates of 8 or more breaks per metaphase, and red arrows highlight pivotal rates of 10 or higher. Cell lines are the same as in A. Fifty metaphases were analyzed per experiment. (C) Cell-cycle analysis by flow cytometry. Exemplary individual measurements. Black histograms are from untreated cultures, and superimposed gray or gray/red are from cultures exposed to MMC (10 ng/mL, 48 hours). The percentage of cells in the G₂ phase is indicated. Red coloring and arrows denote an increased G₂ compartment size. Variability and significance of G₂ phase arrest in repeated measurements are shown in Supplemental Figure 4A. Cell lines are the same as in A. (D and E) Dose-response (survival) curves of parental and mock FAAP100^WT or FAAP100^T542P transduced 1176 cells from cultures exposed to different concentrations of MMC (D) or CDDP (E) for 8 days. Data indicate the mean ± SD of 3 independent experiments. Cell lines are the same as in A and are identical in D and E. FA-B and non-FA are FA and normal control, respectively. LC₅₀, 50% lethal concentration. Note that the transduction of FAAP100^WT complements the repair defect in all assays, whereas FAAP100^T542P does not.

FANCD2 monoubiquitylation capacity of FAAP100-inactivated human and avian cell lines.

(A) FAAP100^T542P mutant 1176 cells (parental) lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. FANCD2 monoubiquitylation was restored only in 1176+FAAP100^WT transduced cells (◄D2-Ub), but not in 1176+FAAP100^T542P or mock transduced cells. FA-L was a monoubiquitylation-deficient FA control. (B) FAAP100^T542-mutant 1176 cells showed reduced levels of FAAP100 protein (see ratio to vinculin in parental and +[empty] vector^pLVX lanes) and lacked FANCD2 monoubiquitylation (◄D2) on immunoblots. 1176+FAAP100^WT- and 1176+FAAP100^T542P-transduced cells both overexpressed FAAP100, but FANCD2 monoubiquitylation was restored only in 1176+FAAP100^WT cells (◄D2-Ub). FA-D2 was a control for absent FANCD2, and non-FA was a monoubiquitylation-proficient normal control. (C) FANCD2 monoubiquitylation was functional in HEK293T cells (not shown) and HEK293T cells mock transfected with the Cas9-containing vector PX459 (◄D2-Ub), but not in the FAAP100-inactivated HEK293T CRISPR/Cas9 clone Cr10 (parental) (◄D2), where it was rescued by transduction with FAAP100^WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (D) FANCD2 monoubiquitylation was functional in parental HAP1 cells (◄D2-Ub), but not in FAAP100-inactivated HAP1 FAAP100^R149Vfs*2 cells (◄D2), where it was restored by transduction with FAAP100^WT, resulting in overexpression of FAAP100 (◄D2-Ub), as shown by FANCD2 and FAAP100 immunoblots. (E) FANCD2 monoubiquitylation was functional in parental DT40 cells (◄D2-Ub), but absent in ΔFAAP100-DT40 cells (◄D2). In the latter, it was rescued by the expression of EGFP-FAAP100^WT (◄D2-Ub), but not by EGFP-FAAP100^T547P (◄D2), which contained the chicken equivalent T547P of human T542P, as shown by FANCD2 and EGFP immunoblots. Note that the strong overexpression of the FAAP100 protein by lentiviral transduction in B–D, where low- and high-exposure blots are shown, was in marked contrast to the transfection in E. Vinculin or RAD50 or tubulin was used as a loading control on all blots. The FAAP100/vinculin ratios were taken from the high-exposure FAAP100 blot. The FANCD2-Ub/FANCD2 ratios semiquantitatively estimated monoubiquitylation.

Cellular FA phenotype of faap100-KO zebrafish.

(A) Schematic of the CRISPR/Cas9-mediated faap100 gene–KO (si:dkey-57h18.1). (B) Growth curves show slower proliferation of primary cell cultures from faap100^–/– fish (circles) than from faap100^+/+ fish (squares). The mean ± SD for multiples of the initial cell number of 3 independent subcultures is shown for each time point, with day 0 counts set to 1. ***P < 0.001 (t test) at day 4. Data were exponentially fitted. (C) Metaphase micrographs after exposure of faap100^+/+ or faap100^–/– cell cultures to MMC (2.5 ng/mL, 24 hours). faap100^–/– cells showed markedly increased chromosome breakage, mostly of the chromatid type. Red arrows indicate radials and black arrows other types of breakage. Original magnification, ×1,000. (D) Box plots reflect the proportion of faap100^+/+ (top) or faap100^–/– (bottom) cells with the indicated number of chromosome breaks per metaphase. Single value (♦), median (─), mean (□), IQR (─), minimum (x), and maximum (x) for the number of breaks from 2 independent experiments; blue symbols represent data from untreated cultures, and red symbols represent data from cultures exposed to MMC (2.5 ng/mL, 24 hours). Light gray shading indicates high rates of 8 or more breaks per metaphase, and the red arrow highlights a pivotal rate of 10 or higher. A total of 31–53 metaphases were analyzed per experiment. (E) Flow cytometric cell-cycle analysis of faap100^+/+ (top) and faap100^–/– (bottom) cell cultures without MMC (– MMC) or after exposure to MMC (+ MMC) (5 ng/mL, 48 hours). Exemplary individual measurements are shown. The percentages of cells in G₂ are shown. Red coloring and arrow indicate an increased G₂ compartment size. (F) Dose-response (survival) curves of faap100^+/+ (top) and faap100^–/– (bottom) cells from cultures exposed to different concentrations of MMC. The mean ± SD of triplicates is shown. (G) Homozygous KO of faap100 resulted in complete sex reversal from female to male. The numbers above the bars represent the number of fish in each sex and genotype category.

Characteristics of the Faap100^–/– mouse.

(A) Proportions and numbers of female (red) and male (blue) Faap100^+/+, Faap100^+/–, and Faap100^–/– mouse offspring from heterozygous mating. (B) Faap100^–/– mice show significantly lower birth weight. Box-and-whisker plots: single value (■, ●, ▲), median (─), mean (□), IQR (─), whiskers (–), and range (x). n = 24 (Faap100^+/+), n = 73 (Faap100^+/–), and n = 18 (Faap100^–/–). ***P < 0.001, by 1-way, repeated-measures ANOVA with Tukey’s test (B and C). (C) Shortened nose-to-tail length in Faap100^–/– neonatal mice. n = 23 (Faap100^+/+) , n = 68 (Faap100^+/–), and n = 15 (Faap100^–/–). **P 0.01 and ***P < 0.001. (D) Reduced postnatal weight gain in Faap100^–/– mice. n = 9–30 (Faap100^+/+), n = 10–83 (Faap100^+/–), and n = 6–11 (Faap100^–/–). *P < 0.05 and **P < 0.01, by 2-way, repeated-measures ANOVA with post hoc Tukey’s test (D and E). (E) Slower growth in body length of Faap100^–/– mice. n = 9 (Faap100^+/+), n = 11–42 (Faap100^+/–), and n = 9 (Faap100^–/–). ***P < 0.001. (F) Gonads in Faap100^–/– mice appear dysplastic, unlike normal gonads in Faap100^+/+ mice. Scale bars: 50 μm. (G) Dose-response survival curves of MEFs exposed to CDDP (8 days) show reduced survival of Faap100^–/– cells. Data are the mean ± SD of triplicates. LC₅₀, 50% lethal concentration. (H) Metaphase micrographs after MMC exposure (100 ng/mL, 48 hours) show increased radials (red arrows) and chromatid breaks in Faap100^–/– MEFs. Original micrographs have been magnified approximately ×1,000. (I) Box plots of breaks per metaphase from 2 independent experiments. Red symbols: MMC-treated; blue: untreated. Gray zone = 8 or more breaks; red arrow = 10 or more breaks. n = 50 metaphases/experiment. (J) Cell-cycle analysis reveals G₂-phase arrest in Faap100^–/– MEFs after MMC exposure (10 ng/mL, 48 hours). Exemplary individual measurements. Black histograms: untreated; gray or gray/red overlaid histograms: MMC-treated. The percentage of G₂ cells is indicated. Red coloring and arrow: increased G₂ compartment size. Variability and significance of G₂-phase arrest in repeated measurements are shown in Supplemental Figure 4D.

Ligase activity, interaction, and subcellular localization studies with FAAP100^T542P.

(A) Reconstitution of FANCD2 monoubiquitylation using purified proteins, including HA-ubiquitin, UBE1, UBE2T, and FANCD2-FANCI complex and, as indicated, FAAP100^WT or FAAP100^T542P, FANCB, and/or FANCL. FANCD2 immunoblot: ◄D2-Ub, monoubiquitylated; ◄D2, nonubiquitylated. (B) Quantitation of results from A. FANCD2-Ub divided by total FANCD2 (percentage) indicates the ubiquitylation efficiency. Lane numbers are identical to those in A. Box plots: single value (♦), median (─), mean (□), IQR (─), minimum (x), and maximum (x) from 3 independent experiments. ***P < 0.001, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (C) Titration of FAAP100^WT (blue) and FAAP100^T542P (red). For reaction mixtures, see A, including FAAP100^WT or FAAP100^T542P, FANCB, and FANCL. (D) Quantitation of results from C (see B and C for details). Data indicate the mean ± SD of 3 independent experiments. (E) In mammalian 2-hybrid assays, FAAP100^WT, but not FAAP100^T542P, interacted with FANCB. FAAP100 fused to the activation domain (orange) or the DNA-binding domain (red). Neither direction of FAAP100 fusion directly interacted with FANCL. Box plots: single value (♦), median (─), mean (□), IQR (─), minimum (x), and maximum (x) of 3 independent experiments. Induction factor, multiples of negative control. *P < 0.05 and **P < 0.01, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (F) In mammalian 3-hybrid assays, FAAP100^WT, but not FAAP100^T542P, interacts with FANCL in both fusion directions in the presence of stably overexpressed FANCB. Controls, calculations, and statistical tests are the same as in E. *P < 0.05 and **P < 0.01, by 1-way, repeated-measures ANOVA with post hoc Tukey’s test. (G and H) Co-IPs from cells transfected with FANCB vector and exposed to MMC (40 ng/mL,16 hours). FAAP100^T542P in 1176 cells and FAAP100^L543_S551del in HEK293T clone Cr12 did not interact with FANCB. Transduced FAAP100^WT rescued the pull-down of FAAP100 by FANCB. Vinculin was used as a loading control. (I) On subcellular protein fractionation, FAAP100^T542P in 1176 fibroblasts and FAAP100^L543_S551del in the HEK293T clone Cr12 were not detected in nuclear extracts (NE) or on chromatin (CB). Transduced FAAP100^WT rescuedFAAP100 relocalization. CE, cytoplasmic extracts. Tubulin, YY1, and histone H3 were used as loading controls. Cells were exposed to MMC (40 ng/mL, 16 hours).