Volume 98, Issue 5, Pages 663-673 (September 1999) The Wilms Tumor Suppressor WT1 Encodes a Transcriptional Activator of amphiregulin  Sean Bong Lee, Karen Huang, Rachel Palmer, Vivi B Truong, Doris Herzlinger, Kathryn A Kolquist, Jenise Wong, Charles Paulding, Seung Kew Yoon, William Gerald, Jonathan D Oliner, Daniel A Haber  Cell  Volume 98, Issue 5, Pages 663-673 (September 1999) DOI: 10.1016/S0092-8674(00)80053-7

Figure 1 Induction of amphiregulin mRNA by WT1(−KTS) (A) Northern blot analysis of U2OS cells with tetracycline-regulated expression of WT1(−KTS), WT1(+KTS), or vector, following growth in the presence or absence (11 hr) of tetracycline. Blots were hybridized with probes for WT1, amphiregulin, and GAPDH (loading control). (B) Kinetics of WT1 and amphiregulin induction following tetracycline withdrawal. Total RNA was isolated following growth in the presence of tetracycline and 0, 3, 6, or 9 hr following drug withdrawal. (C) Northern blot analysis of embryonic rat kidney RSTEM cells with tetracycline-regulated expression of WT1(−KTS) and WT1(+KTS). Total RNA was isolated 6 hr after tetracycline withdrawal and probed for WT1, amphiregulin, and GAPDH. Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)

Figure 2 Identification of WT1-Responsive Element within amphiregulin Promoter (A) Schematic representation of the human amphiregulin promoter indicating the relative positions of the TATA box (−238 to −233), CRE site (−274 to −267), and adjacent WRE (−292 to −283; see below). Nucleotide numbers refer to promoter sequence (Plowman et al. 1990). Three luciferase reporter plasmids (A–C) generated in the promoterless backbone pGL2 are shown, representing nested deletions. (B) Activation of the amphiregulin promoter by WT1(−KTS). Luciferase activity, relative to vector-transfected cells, was measured in NIH3T3 fibroblasts 48 hr following cotransfection of reporter constructs (2 μg) and either WT1(−KTS) or WT1(+KTS) expression plasmids (5 μg). Reporter plasmids are the nested deletion contructs pGL2-A-C, in addition to the respective constructs with deletion of the CRE site (ΔCRE), to demonstrate the small and nonsynergistic effect of this site adjacent to the WRE. Transfection efficiency was standardized using a cotransfected reporter (human growth hormone), and equal amounts of CMV promoter were present in each transfection; standard deviations were derived from three independent experiments. (C) DNase I footprint analysis of a 300 bp fragment of the amphiregulin promoter (nucleotides −385 to −87). The end-labeled fragment was incubated with 0, 20, or 80 μl of GST-WT1(−KTS) or GST-WT1(+KTS), followed by partial DNase I digestion. The 16 bp region protected by 80 μl of WT1(−KTS) is indicated by the black box (−294 to −278); the CRE site is represented by a white box. (D) EMSA analysis of amphiregulin promoter fragments following incubation with the zinc finger domains of either WT1(−KTS) or WT1(+KTS). Overlapping genomic probes were generated by restriction digestion of the pGL2-B promoter fragment using enzyme pairs XhoI/AatII and NarI/AatII, whose cleavage sites are indicated. The black box indicates the DNase I–protected region. End-labeled probes (free probe designated P) were incubated with 200 ng of the respective GST fusion proteins. The positions of unbound (bracket) and complexed probe (arrow) are shown. Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)

Figure 3 Characterization of WT1-Responsive Element within the amphiregulin Promoter (A) Minimal WRE (10 nucleotides), identified by EMSA, following thymidine substitution of the 5′ and 3′ terminal residues of the 16 nucleotide fragment protected by DNase I footprint analysis (shown at top in antisense orientation). Complex formation by WT1(−KTS), but not WT1(+KTS), is shown for the full DNase I–protected fragment (at right). Thymidine-substituted residues are indicated in boxes (sub), free probe is denoted with a bracket, and the position of the protein complex is indicated with an arrow. The G→T change at the 3′ end of WRE (as), which results in increased binding by WT1(−KTS), represents the tenth position of the WRE (see below). (B) Identification of essential residues within the WRE. The ten nucleotides of the Amphiregulin antisense WRE (as) are aligned with the ten nucleotides of the sense WTE (s), the optimized in vitro binding sequence for WT1(−KTS) (Nakagama et al. 1995). Identical residues are marked by a vertical line. EMSA analysis of WT1(−KTS), WT1(+KTS), or free probe (P) for the WRE (left) is compared with probes containing a substitution at each nucleotide constituting the WRE. All nonthymidine residues were substituted to thymidine; thymidine residues were substituted to adenosine. Numerical positions correspond to the 5′ to 3′ WRE (as) and WTE (s) sequences. Equal amounts of probe and WT1 protein were added in all cases; both parts of the panel were derived from the same experiment. Migration of free probe (bracket) and the protein complex (arrow) are shown. (C) Relative binding affinity of WT1(−KTS) for WRE, WTE, and substituted WRE (G6T, G5T, and T8A), assessed by competition of unlabeled probes (indicated above each lane; 300- or 600-fold molar excess) with the end-labeled WRE probe. Incubation of WT1(−KTS) or WT1(+KTS) with WRE, in the absence of competing probes, is shown at left. Migration of the protein complex is denoted by arrow. (D) Effect of mutant WRE on transcriptional activation of the amphiregulin promoter by WT1(−KTS). Luciferase activity, relative to vector transfection, following cotransfection of NIH3T3 cells with CMV-driven WT1(−KTS) or WT1(+KTS) and the following reporters: full-length amphiregulin promoter (pGL2-A), full-length promoter containing point mutations disrupting the WRE (adenosine substitutions at positions T4, G6, and G7; pGL2-A-mWRE), and a truncated promoter containing only the CRE site and TATA box (pGL2-C). Results are shown for two independent transfection experiments. Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)

Figure 4 Colocalization of WT1 and amphiregulin Expression in Structures of the Differentiating Kidney Immunohistochemical analysis of (A) WT1 and (B) Amphiregulin expression in differentiating structures within the nephrogenic zone of the developing human kidney (18 weeks). Blastema (B) denotes the condensed mesenchymal cells located in the outermost subcortical region and shows low levels of expression. Developing glomeruli (DG), with peak WT1 and Amphiregulin expression, are located centrally to the blastemal layer. Mature glomeruli (MG) are in the most central region of the nephrogenic zone. Insets show strong expression of both WT1 and Amphiregulin in the podocyte layer of developing glomeruli. RNA in situ analysis of (C) WT1 and (D) amphiregulin expression in sections of the 18-week human kidney. Blastema, developing, and mature glomeruli are identified as above. Specificity of the hybridization was confirmed through the use of sense probes (not shown). Insets show high magnification to illustrate increased expression in condensed blastema for both WT1 and amphiregulin. Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)

Figure 5 Induction of Ureteric Bud Branching by Recombinant Amphiregulin in Cultured Mouse Metanephric Kidney Rudiments Staining of the ureteric bud network (FITC-Dolichos Bifloris stain) in microdissected mouse metanephric kidney rudiments, cultured in the absence or presence of 20 ng/ml purified Amphiregulin. A representative section is shown in upper panel. Dose dependence of the effect of Amphiregulin (0, 5, 20, or 100 ng/ml) on branching of single ureteric bud tubules was quantitated (lower panel). For each culture condition, a minimum of 15 rudiments were analyzed and the number of terminal branches recorded. Significant differences in ureteric bud branching were detected at Amphiregulin concentrations greater than 5 ng/ml (Student t-test; p < 0.001). Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)

Figure 5 Induction of Ureteric Bud Branching by Recombinant Amphiregulin in Cultured Mouse Metanephric Kidney Rudiments Staining of the ureteric bud network (FITC-Dolichos Bifloris stain) in microdissected mouse metanephric kidney rudiments, cultured in the absence or presence of 20 ng/ml purified Amphiregulin. A representative section is shown in upper panel. Dose dependence of the effect of Amphiregulin (0, 5, 20, or 100 ng/ml) on branching of single ureteric bud tubules was quantitated (lower panel). For each culture condition, a minimum of 15 rudiments were analyzed and the number of terminal branches recorded. Significant differences in ureteric bud branching were detected at Amphiregulin concentrations greater than 5 ng/ml (Student t-test; p < 0.001). Cell 1999 98, 663-673DOI: (10.1016/S0092-8674(00)80053-7)