The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription factor which is expressed in a tissue-specific manner. Our studies indicate that in addition to the promoter, other regulatory elements are required for tissue-specific expression of this gene. A 258-base pair hematopoietic specific enhancer in intron 3 of the WT1 gene increased the transcriptional activity of the WT1 promoter by 8 -10-fold in K562 and HL60 cells. Sequence analysis revealed both a GATA and a c-Myb motif in the

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... ancer fragment. Mutation of the GATA motif decreased the enhancer activity by 60% in K562 cells. Electrophoretic mobility shift assays showed that the GATA-1 protein in K562 nuclear extracts binds to this motif. Cotransfection of the enhancer containing reporter construct with a GATA-1 expression vector showed that GATA-1 transactivated this enhancer, increasing the CAT reporter activity 10 -15-fold. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT reporter construct and a c-Myb expression vector showed that c-Myb transactivated the enhancer by 5-fold. A DNase I-hypersensitive site has also been mapped in the 258-base pair enhancer region. These data suggest that GATA-1 and c-Myb are responsible for the activity of this enhancer in hematopoietic cells and may bind to the enhancer in vivo.