Transactivation of an intronic hematopoietic-specific enhancer of the human Wilms' tumor 1 gene by GATA-1 and c-Myb.
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 enhancer 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.
Zhang
X
X
Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Xing
G
G
Fraizer
G C
GC
Saunders
G F
GF
D001483:Base Sequence; D019070:Cell Lineage; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D016162:Genes, Wilms Tumor; D018922:HL-60 Cells; D006801:Humans; D007438:Introns; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000473:pathology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D014157:Transcription Factors; D003850:Deoxyribonuclease I