Binding of the Wt1(–KTS) isoform to the minimal human EPO promoter. (A) EMSA demonstrating binding of the Wt1(–KTS) protein to a DNA fragment (oligo –66/–37) of the minimal human EPO promoter. Arrowhead indicates the specific retardation band. The binding oligonucleotide (for detailed base pair sequence, see Table 1) harbors nt –66 to –37 relative to the transcription start site of the EPO gene. Note that the specific retardation signal (lane 8) can be competed with a 5-fold (lane 10) and a 100-fold (lane 11) molar excess of a 21-bp DNA sequence containing the previously identified Wt1(–KTS) binding site in the vitamin D receptor gene promoter. For comparison, Wt1(+KTS) protein binds to the same oligo –66/–37 with much lower affinity (lane 9). Introducing different mutations (mutants A, B, C; Table 1) into oligo –66/–37 reduces Wt1(–KTS) binding. Note that both Wt1 proteins (±KTS) failed to interact with 2 other GC-rich oligonucleotides (oligo –116/–87 and oligo –86/–67) of the human EPO promoter. EMSA experiments were performed with 20 μg recombinant Wt1 protein in each reaction. (B) Chromatin-immunoprecipitation analysis of protein interactions with the EPO gene. Note illustration of the potential binding sites of Sp1, GATA, and Wt1 transcription factors within the minimal EPO promoter, relative to the transcription start site (arrow) and the first exon (Ex I). Sizes of the DNAfragments obtained in Hep3B cells after sonification were revealed by electrophoresis in an ethidium bromide–stained 1.6% agarose gel. PCR-amplified products of the immunoprecipitates were electrophoresed in a 1.5% agarose gel and stained with ethidium bromide; for better visualization, the gel photograph is presented as a negative of the original. Apparently, under normoxia, Wt1 and Sp1 bind to the 5′ promoter but not to the hypoxia-responsive 3′ enhancer of the EPO gene.