Figure 1.
UTX is required for cell survival following IR exposure in Kc cells.
(A) Cell counts at 2 and 4 days following RNAi treatment are indicated. Note that at 2 days, there is no difference in the growth rate of utx RNAi-treated compared to control (Ctrl) RNAi-treated cells. The asterisk indicates P<0.05 compared with the Ctrl RNAi group on the same day, as determined by two-way ANOVA followed by a post-hoc Tukey’s test for comparisons between groups. (B) utx RNAi-treated cells display significantly reduced cell counts compared to control RNAi-treated cells and over-expression wild type UTX could rescue RNAi effect but not mutant UTX after IR exposure. 4 days RNAi-treated cells were irradiated at doses of 4 and 8 Gy and examined relative cell viability after 2 days.(C) Western blot analysis of UTX expression confirming the efficiency of utx RNAi-mediated knockdown and over-expression of UTX. (D) IR treatment causes no detectable change in UTX protein levels. β-Tubulin (Tub) levels were used as loading controls. All of the data are representative of at least three independent experiments with similar results. Error bars indicate standard deviations from triplicate sets of the presented experiment.
Figure 2.
UTX is required for the expression of ku80 after IR exposure via the demethylation of H3K27me3 in Kc cells.
(A) qRT-PCR analysis of the mRNA expression of the indicated genes before and after IR exposure in RNAi-treated Kc cells. The relative expression levels are normalized to β-tubulin levels. Note that ku80 is the only gene that requires utx for its expression. (B) qRT-PCT analysis of ku80 expression in different treatment cell. Over-expression WT UTX could rescue ku80 expression in UTX RNAi cell after IR but not mutant UTX. (C) ChIP assay with an anti-UTX antibody and the ku80 promoter with and without IR. Note the dramatic increase in the UTX occupancy of the ku80 promoter after IR. (D) ChIP assay for H3K27me3 at the ku80 promoter in different treatment Kc cells after IR. (E) The diagrams show PCR-amplified regions (double arrows) relative to the first exons (black box) in the ChIP analysis of the three DNA repair genes ku80, ku70 and mre11 and a control gene, panier (pnr). (F) Changes in H3K27me3 levels at the indicated genes 2 hours after IR treatment with a dose of 8 Gy. The H3K27me3 levels in the promoter regions of those genes were determined via ChIP assays and compared to the input genomic DNA.
Figure 3.
p53 and UTX are recruited in an interdependent manner to the ku80 promoter region.
(A) ku80 expression following IR exposure in Kc cells subjected to RNAi treatment, as indicated. (B, C) ChIP analysis of the physical occupancy of p53 and UTX at the ku80 promoter region. Note that knockdown of utx eliminates the increase in p53 binding, and knockdown of p53 reduces the binding of UTX to the ku80 promoter. (D) ChIP assay for H3K27me3 at the ku80 promoter in Kc cells treated with control or utx RNAi after IR. (E) Coimmunoprecipitation was performed using anti-p53 and anti-UTX antibodies and whole cell extracts of Kc cells. The immunoprecipitates were subjected to Western blot analysis with the indicated antibodies. (F, G) Western blot analysis to confirm the knockdown efficiency of p53 RNAi. β-Tubulin (β-Tub) levels were used as a loading control.
Figure 4.
UTX is required for the expression of ku80 following IR exposure in Drosophila.
(A) Schematic illustration of the gene structure of wild type utx and a utx mutant allele (utxΔ95) generated via the imprecise excision of a P-element insertion. (B, C) Genomic PCR (B) and Western blot (C) analyses to verify the utxΔ95 genotype. (B) An approximately 250-bp band is detected in adult flies with a utxΔ95/Cyo genotype, but absent from the w1118 genotype. For details, please see the Materials and Methods section. (C) An approximately 130-kDa band indicated by an arrow in w1118 flies was not detected in the utxΔ95/utx1 third instar larvae. A non-specific band is indicated by an asterisk, and β-Tubulin (β-Tub) was used as a loading control. (D) qRT-PCR analysis of mRNA expression for the indicated genes before and after IR exposure in the w1118 and utxΔ95/utx1 third instar larvae. The relative expression levels are normalized to β-tubulin levels. Note that ku80 is the only gene that requires utx for its expression.
Table 1.
The hatching rate of Drosophila embryo.
Table 2.
The hatching rate of Drosophila embryo.
Figure 5.
UTX is not responsible for the upregulation of apoptosis-related genes following IR exposure.
(A, B) qRT-PCR analysis of the mRNA expression of the indicated genes before and after IR treatment in RNAi-treated Kc cells, as shown for w1118 and utxΔ95/utx1 third instar larvae. The relative expression levels are normalized to β-tubulin levels. (C) The diagrams show PCR-amplified regions (double arrows) relative to the first exons (black box) in the ChIP analysis for two apoptosis-related genes, hid and rpr. The changes in H3K27me3 levels at the genes are indicated 2 hours after IR treatment with a dose of 8 Gy.
Figure 6.
A model for the regulation of DNA damage response genes associated with DNA double-strand breaks in cells is suggested based on the results presented in Figures 2–5.
See text.