75 Tissue-Specific Activation of the Erythropoietin Gene by the Wilms' Tumor Suppressor Wt1(-KTS)

Abstract

The mechanisms of the developmental-stage and tissue-specific regulation of erythropoietin (Epo) expression under normoxia are only partially understood yet. The Wilms tumor suppressor gene, Wt1, encodes a zinc finger transcription factor, which is required for the normal development of various organs, including the kidney, heart, and eye. Recent findings suggest a role of Wt1 in the development of the hematopoietic system. In our study, we tested the hypothesis that Wt1 is involved in Epo gene regulation. Using electrophoretic mobility shift assay and in vitro DNA-footprinting, we identified the binding of the Wt1(-KTS) variant, which acts as a transcription factor, to the minimal Epo promoter. Under normoxia, Epo mRNA and protein expression were significantly upregulated in human embryonic kidney HEK 293 cells, which had been stably transfected with Wt1. A reporter plasmid construct harboring the 117bp minimal human Epo promoter was activated more than 20-fold by the Wt1(-KTS) isoform in transient transfection assays. Mutations in the Wt1 binding site in the Epo promoter did not interfer with the hypoxic induction of reporter constructs harboring the downstream Epo enhancer. Hepatic Epo mRNA expression was significantly decreased in Wt1 −/− mouse embryos (e12.0). Immunohistochemical fluorescence-double-staining indicated also the co-expression of Wt1 and Epo in neuronal cells of the developing dorsal root ganglion. Co-expression has been also found in the adult testis were both genes may have antiapoptotic function. Preliminary data indicate furthermore that Wt1(-KTS) modulates the binding of other transcription factors, such as Sp1, to the Epo promoter. In conclusion, Wt1 is a novel transcriptional activator in the developmental-stage and tissue-specific regulation of Epo.