Fig 2: Regulation of DPF3 expression and effect of DPF3 on tubular cell growth.A, relative expression levels of DPF3 in primary renal tubular cells exposed to 1 mM of DMOG for 16 h. Samples were stratified according to the rs4903064 genotype (TT = 42, CT/CC = 44). Values were normalized to expression levels of the housekeeping gene HPRT and to values from corresponding untreated cells. Each dot represents the mean value from cells of one individual. qPCR was performed in duplicates per individual. Bars indicate mean values ± SD. Student’s t test, ∗∗∗∗p < 0.0001. B, DPF3 expression levels in isolated tumor cells compared to corresponding untreated tubular cells from 8 (TT) and 14 (CT/CC) individuals, respectively. Bars indicate mean values ± SD. One sample t test, ∗p < 0.05. C, top, allelic ratios of rs4903064 in cells from heterozygous individuals exposed to DMOG 1 mM or left untreated. One sample t test, ∗∗∗ p < 0.001. Bottom, allelic ratios of rs4903064 in ccRCC cells with high (CA9+) or low (CA9−) CA9 expression as a marker for HIF-1α activity. Values are mean from technical triplicates, and each dot represents values derived from one individual. Bars indicate mean values ± SD. t test, ∗ p < 0.05. D, Western blot for DPF3 and beta actin from lysates of different clones of HKC-8 cells with or without defective DPF3. E, Western blot for DPF3 and beta actin from lysates of two different huPTC lines transfected either with nontargeting (nt) guides or guides targeting DPF3. F, proliferation assays of the clones of cells indicated in (D). Dark lines and dots indicate the mean value from the three different clones of cells, which were tested in biological triplicates. Light gray and light blue lines indicate individual values for the three different clones of cells. G, proliferation assay from cells indicated in (E). H, Western blot for DPF3 and beta actin from lysates of primary renal tubular cells (PTC) transfected either with nontargeting (nt) guides or guides targeting DPF3. Cells were harvested at day 3 after transfection. I, cyst volume of PTC 7 days after plating in a Matrigel matrix (n = 3 wells per condition, 4 quadrants per well). J, representative images of the cysts in the Matrigel matrix. Values are mean ± SD. t test, ∗p < 0.05, ∗∗p < 0.01 for F, G, and I. CA9, carbonic anhydrase 9; ccRCC, clear cell renal cell carcinoma; DMOG, dimethyloxalylglycine; DPF3, double PHD fingers 3; huPTC, human urinary primary tubular cells.

Fig 3: Expression of DPF3 in renal cancer.A, relative expression levels of DPF3 compared to corresponding normal tissue in 111 clear cell (ccRCC), 15 papillary (pRCC), and 11 chromophobe (chRCC) tumors. Mean fold change is indicated above the values for each tumor entity. One sample t test versus values from normal tissue, ∗∗p < 0.01, ∗∗∗p < 0.001. Bars indicate mean fold change ± SD. B, immunostaining for DPF3 in the different renal tumor subtypes indicates increased nuclear staining for DPF3 in ccRCC. C, immunoreactivity scores for 306 clear cell, 44 papillary, and 22 chromophobe tumors. Bars indicate median, error bars are the 95% confidence interval. Mann–Whitney test, ∗p < 0.05. D, relative mRNA expression levels of DPF3 in ccRCC samples from (A) stratified for the rs4903064 genotype (T = protective, C = risk allele). Bars indicate mean fold expression ± SD. One-way ANOVA, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. E, allelic ratio for rs4903064 in pre-mRNA isolated from the indicated tissues (n = 7 for ccRCC, n = 6 for non-ccRCC). Each dot represents the mean of one individual determined in triplicates. Bars indicate mean values ± SD. ∗, t test, p < 0.05. F, immunoreactivity score for DPF3 in a selection of ccRCC sections from (C) stratified according to the rs4903064 genotype (TT n = 34, CT n = 22, CC n = 6). Bars indicate median, error bars are the 95% confidence interval. Mann–Whitney test, ∗∗∗p < 0.001. ccRCC, clear cell renal cell carcinoma; DPF3, double PHD fingers 3.

Fig 4: HIF binding to the RCC risk allele.A, HIF ChIP qPCR results for the DPF3 locus. Experiments were performed from cells isolated from 11 TT, 11 CT, and 1 CC individuals. Bars indicate mean values ± SD. Student’s t test, ∗p < 0.05, ∗∗p < 0.01. B, binding of HIF to the control locus at EGLN3 is not affected by the rs4903064 genotype. Bars indicate mean values ± SD. C, HIF ChIP-seq tracks in two different ccRCC cell lines at the rs4903064-associated enhancer (TT: RCC4 and CC: individual 40,911,432 from Yao et al.). HIF binding is only detectable in cells from a patient carrying the C allele (red, HIF-1α: 12 × C from 12 reads in total, HIF-2α 99 × C from 99 reads in total). D, same tracks as in (C) covering the EGLN3 control locus. E and F, H3K27ac ChIP-seq tracks from corresponding normal (blue) and ccRCC (red) tissue indicates increased activity of the disease-associated regulatory element at DPF3 (E, highlighted in yellow) and of the EGLN3 locus (F) in tumors. G, H3K27ac ChIP-seq tracks for two different ccRCC cell lines at the rs4903064-associated enhancer (TT: 786-O and CC: individual 40,911,432 from Yao et al.). Levels of the active enhancer mark H3K27ac are high in the individual carrying the C allele and reduced upon VHL re-expression. H, same tracks as in (G) covering the EGLN3 control locus. Note that H3K27ac levels are reduced upon VHL re-expression in both cell lines. ccRCC, clear cell renal cell carcinoma; ChIP, chromatin immunoprecipitation; DPF3, double PHD fingers 3; EGLN3, Egl-9 family hypoxia inducible factor 3; HIF, hypoxia-inducible transcription factor; KIRC, kidney renal clear cell carcinoma; SNP, single nucleotide polymorphism; VHL, von Hippel-Lindau.