Article Figures & Data
Figures
- Fig 1
Homozygous inactivation of murine Uaf1 causes embryonic lethality. (A) Map of the genomic Uaf1 gene, showing disruption by a gene trap vector. The gene trap vector was designed in three parts: SA (the splice acceptor sequence), a Trap cassette (the selection and reporter construct, a neo selection), and pA (the polyadenylation signal). The gene trap vector was inserted in intron 2 (between exon 2 and exon 3) of the Uaf1 gene. The locations of WT and KO primers for PCR genotyping are shown. (B) Viability of Uaf1-deficieint mice at various stages of development. (C) Representative images of Uaf1+/+ and Uaf1−/− embryos at E7.5. (D) Appearance of Uaf1+/− and Uaf1+/+ mice at birth. (E) Skeletal staining of 3.5-week-old Uaf1+/− and Uaf1+/+ mice. (F) Growth curve of Uaf1+/− and Uaf1+/+ mice (n = 30) from the newborn stage to 49 days after birth. *, P < 0.001. The error bars indicate standard deviations of 30 mice. (G) Representative photographs of testes from 12-week-old Uaf1+/− and Uaf1+/+ male mice. (H) Comparison of the testis size and oocyte numbers in Uaf1+/− and Uaf1+/+ mice. Testes were obtained from 12-week-old male Uaf1+/− and Uaf1+/+ mice (n = 20). For the quantitative analysis of oocyte numbers, visible oocytes were counted per section, and the numbers of oocytes in 12-week-old female Uaf1+/− and Uaf1+/+ mice (n = 20) were determined for each section; 7 sections per female were analyzed. *, P < 0.001. (I and J) Histology of the testes and ovaries from two representative 12-week-old Uaf1+/− and Uaf1+/+ mice. H&E staining of tissue sections of ovaries and testes is shown (scale bar, 200 μm). The arrows indicate smaller seminiferous tubules in the testes and smaller numbers of oocytes in the ovaries. (C to J) Representative analyses; n = 4 to 20 for each genotype.
- Fig 2
Uaf1+/− MEFs are hypersensitive to DNA-damaging agents. (A) Immunoblots of the lysates from primary Uaf1+/− and Uaf1+/+ MEFs. Usp1 antibody against the C-terminal epitope of Usp1 was used for immunoblots. (B to F) Survival plots of Uaf1+/− and Uaf1+/+ MEFs exposed to various DNA-damaging agents. The MEFs were exposed to MMC, UV, IR, etoposide, or AZD 2281, and survival was determined using a colony assay. P < 0.005 for Uaf1+/− versus Uaf1+/+ MEFs. (G to I) The Uaf1+/− and Uaf1+/+ MEFs were exposed to MMC for 48 h, and metaphase spreads of chromosomes were scored for chromosomal abnormalities. (G and H) Aberrations per cell (G) and percentages of cells with radials (H). (I) Representative images of metaphase spreads of the chromosomes. The arrows indicate radials in Uaf1+/− MEFs upon MMC exposure. The data in panels B to H are representative of two independent experiments in triplicate, and means ± standard errors are shown.
- Fig 3
Uaf1-deficient cells are defective in HR repair. (A) Defective HR repair in Uaf1+/− MEFs. Uaf1+/− and Uaf1+/+ MEFs harboring DR-GFP reporter were infected with retroviruses encoding hemagglutinin (HA)-tagged I-SceI or vector. The GFP expression was then determined after 96 h using flow cytometry, and the GFP expression as a readout of HR activity is shown. The data are from three Uaf1+/+ and four Uaf1+/− MEF lines. P < 0.001 for Uaf1+/− versus Uaf1+/+ MEFs after I-SceI. (B) Immunoblots of lysates from Uaf1+/− and Uaf1+/+ MEFs showing equal expression of HA-tagged I-SceI. (C) Defective HR repair in U2OS cells with knockdown of Uaf1. U2OS cells with DR-GFP reporter were transfected with siRNAs against UAF1, USP1, USP12, USP46, or BRCA2 (as a positive control), followed by transfection with I-SceI reporter plasmid. The GFP expression was then determined after 48 h using flow cytometry. The GFP expression as a readout of HR activity is shown. Two independent experiments were performed in triplicate, and means and standard errors are shown. (D) Survival curves of U2OS cells exposed to ABT-888. U2OS cells were transfected with siRNAs against control (LacZ), UAF1, USP1, USP12, USP46, or FANCD2 (as a positive control) and exposed to ABT-888. Five days after the exposure, the cell viability was determined. Two independent experiments were performed in triplicate, and means ± standard errors are shown. (E) Immunoblots of the lysates from U2OS cells transfected with control siRNA or siRNA against UAF1, USP1, or BRCA2 are shown on the left. qPCR analysis of RNA from U2OS cells transfected with control siRNA or siRNA against USP12 and USP46 is shown on the right.
- Fig 4
Uaf1−/− mESCs are hypersensitive to DNA-damaging agents. (A) Immunoblots of the lysates from Uaf1−/− or Uaf1+/+ mESCs. Usp1 antibody against the C-terminal epitope of Usp1 was used for the immunoblots. (B to E) Survival plots of Uaf1−/−, Uaf1+/−, and Uaf1+/+ mESCs exposed to various DNA-damaging agents. mESCs were plated in gelatin-coated 6-well plates without feeder cells and exposed to MMC, UV, IR, or acetaldehyde. The cells were then cultured for 2 weeks, and the colonies were fixed with methanol, stained with crystal violet, and counted. P < 0.001 for Uaf1−/− versus Uaf1+/+ mESCs. (F to H) The Uaf1−/− and Uaf1+/+ mESCs were exposed to MMC for 48 h, and metaphase spreads of chromosomes were scored for chromosomal abnormalities. (F) Number of aberrations per cell. (G) Percentages of cells with radials. (H) Representative images of metaphase spreads of the chromosomes. The arrow indicates radials in Uaf1−/− mESCs upon MMC exposure. The data in panels B to G are representative of the two independent experiments in triplicate, and means ± standard errors are shown.
- Fig 5
Uaf1-deficient mESCs exhibit increased Fancd2 monoubiquitination and decreased Fancd2 nuclear foci. (A) Immunoblots of the lysates from Uaf1−/− and Uaf1+/+ mESCs exposed to DNA-damaging agents. Usp1 antibody against the C-terminal epitope of Usp1 was used for immunoblots. (B and C) Fancd2 or 53BP1 foci in Uaf1−/− and Uaf1+/+ mESCs 3 h after IR (10 Gy) exposure. Quantification of cells with more than 5 foci/cell representing at least 100 nuclei each in three independent experiments was carried out. *, P < 0.001 for Uaf1−/− and Uaf1+/+ mESCs after IR exposure. Means and standard errors are shown in panels B and C.
- Fig 6
Uaf1 deficiency results in decreased Id1 expression. (A) Skeletal staining of Uaf1+/− and Uaf1+/+ E19 embryos. The arrow indicates the location of the femur. (B) Immunoblots of the lysates from femurs (in panel A) of Uaf1+/− and Uaf1+/+ E19 embryos. (C to E) Uaf1−/− and Uaf1+/+ mESCs were cultured without LIF in order to promote differentiation. RNA was then isolated at days (D) 0, 2, 4, 6, and 8 for quantification of Id1 and Uaf1 by qPCR. Id1 or Uaf1 expression upon differentiation of Uaf1−/− and Uaf1+/+ mESCs is shown. The data are shown as relative expression of Id1 or Uaf1 mRNA in Uaf1−/− mESCs compared to the wild-type controls. The samples were normalized using GAPDH expression.
- Fig 7
Uaf1 deficiency causes decreased cell proliferation and inhibits tumorigenesis. (A) Uaf1+/− mice have decreased TPA-induced epithelial proliferation. Six-week-old Uaf1+/− and Uaf1+/+ mice were exposed topically to TPA for 4 weeks. The mice were sacrificed, and the skin histology was evaluated by microscopy. H&E staining of tissue sections of skin (representative analysis; n = 4 for each genotype) is shown. The scale bar represents 100 μm, and the arrows indicate epithelial layers. (B to E) UAF1 knockdown in lung cancer cells suppresses growth in soft agar and reduces tumorigenesis in nude mice. (B) The cDNA encoding a UAF1 shRNA or shScramble (Vector) was stably expressed in the RAS-driven human lung adenocarcinoma epithelial cell line A549. The cells were subjected to growth assay or analyzed for tumorigenicity in nude mice. Immunoblots of the A549 cell lysates are shown. (C) Usp1 antibody against the N-terminal epitope of Usp1 was used for immunoblots. A549 cells were plated in soft agar, and transformed foci were counted (at ×100 magnification) after 2 weeks in culture. The data are means ± standard errors of three independent experiments. Each experiment was performed in triplicate plates (P < 0.001; t test). (D and E) Tumorigenicity of A549 cells in xenograft mouse models. UAF1 shRNA- or shScramble-transfected A549 cells were injected subcutaneously as xenografts in nude mice (two-site injections), and every 3 days the tumor volume was measured. The data represent the average tumor size for 10 mice in each group (more than 10 tumors, due to the two-site injections; P < 0.001). Quantification of the tumor volume of the xenografts at day 35 is shown in panel E. The data are means ± standard errors.
