TRANSACTIVATION OF AN INTRONIC HEMATOPOIETIC-SPECIFIC ENHANCER OF THEHUMAN WILMS-TUMOR-1 GENE BY GATA-1 AND C-MYB

The Wilms' tumor 1 gene (WT1) encodes a zinc-finger transcription fact or which is expressed in a tissue-specific manner. Our studies indicat e that in addition to the promoter, other regulatory elements are requ ired for tissue-specific expression of this gene, A 258-base pair hema topoietic 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 i n the enhancer fragment. Mutation of the GATA motif decreased the enha ncer activity by 60% in K562 cells. Electrophoretic mobility shift ass ays showed that the GATA-1 protein in K562 nuclear extracts binds to t his motif. Cotransfection of the enhancer containing reporter construc t with a GATA-1 expression vector showed that GATA-1 transactivated th is enhancer, increasing the CAT reporter activity 10-15-fold. Similar analysis of the c-Myb motif by cotransfection with the enhancer CAT re porter construct and a c-Myb expression vector showed that c-Myb trans activated the enhancer by B-fold. A DNase I-hypersensitive site has al so been mapped in the 258-base pair enhancer region, These data sugges t that GATA-1 and c-Myb are responsible for the activity of this enhan cer in hematopoietic cells and may bind to the enhancer in vivo.