Repression of the c-myb gene by WT1 protein in T and B cell lines.
The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.
McCann
S
S
Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center, Stanford, California, USA.
Sullivan
J
J
Guerra
J
J
Arcinas
M
M
Boxer
L M
LM
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051766:Early Growth Response Protein 1; D016162:Genes, Wilms Tumor; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011519:Proto-Oncogenes / Q000187:drug effects*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D025721:WT1 Proteins
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D004247:DNA