WT1 encodes a zinc finger transcription factor implicated in kidney differentiation and tumorigenesis. In reporter assays, WT1 represses transcription from GC- and TC-rich promoters, but its physiological targets remain uncertain. We used hybridization to high-density oligonucleotide arrays to search for native genes whose expression is altered following inducible expression of WT1. The major target of WT1 was amphiregulin, a member of the epidermal growth factor family. The WT1(-KTS) isoform binds directly to the amphiregulin promoter, resulting in potent transcriptional activation. The in vivo expression profile of amphiregulin during fetal kidney development mirrors the highly specific pattern of WT1 itself, and recombinant Amphiregulin stimulates epithelial branching in organ cultures of embryonic mouse kidney. These observations suggest a model for WT1 as a transcriptional regulator during kidney differentiation.

The GnRH gene is exclusively expressed in a discrete population of neurons in the hypothalamus. The promoter-proximal 173 bp of the rat GnRH gene are highly conserved through evolution and are bound by multiple nuclear proteins found in the neuronal cell line, GT1-7, a model for the GnRH-expressing hypothalamic neuron. To explore the protein-DNA interactions that occur within this promoter and the role of these interactions in targeting GnRH gene expression, we have mutagenized individual binding sites in this region. Deoxyribonuclease I protection experiments reveal that footprint 2, a 51-bp sequence that confers a 20-fold induction of the GnRH gene, is comprised of at least three independent protein-binding sites. Transfections of the GnRH promoter-reporter plasmid containing a series of block mutations of footprint 2 into GT1-7 neurons indicate that each of the three putative component sites contributes to transcriptional activity. Mutations in footprint 4 also decrease GnRH gene expression. Footprint 4 and the promoter-proximal site in footprint 2 contain octamer-like motifs, an element that is also present in the neuron-specific enhancer of the rat GnRH gene located approximately 1.6 kb upstream of the promoter. Previous studies in our laboratory have demonstrated that two enhancer octamer sites are bound by the POU-homeodomain transcription factor Oct-1 in GT1-7 cells. We now show that Oct-1 binds the octamer motifs within footprints 2 and 4. Thus, Oct-1 plays a critical role in the regulation of GnRH transcription, binding functional elements in both the distal enhancer and the promoter-proximal conserved region.

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