The WT1 protein is a negative regulator of the normal bcl-2 allele in t(14;18) lymphomas.
The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a potential WT1 binding site in the bcl-2 5'-flanking sequence was identified on the normal silent allele. Electrophoretic mobility shift assays with the bcl-2 WT1 site demonstrated a single specific complex. UV cross-linking and Western analysis revealed that this gel shift complex contained WT1 protein. Deletion or mutation of the WT1 site resulted in an increase in activity of the bcl-2 promoter in DHL-4 cells. Cotransfection with a 3:1 ratio of a WT1 expression vector to the bcl-2 promoter construct led to a 3.0-fold repression of the bcl-2 promoter. Cotransfection with a WT1 expression vector and the bcl-2 promoter with the mutated WT1 site resulted in only 1.2-fold repression. We conclude that the WT1 site functions as a negative regulatory site for the normal silent bcl-2 allele in t(14;18) lymphomas. The WT1 site is not occupied on the translocated bcl-2 allele.
Heckman
C
C
Center for Molecular Biology in Medicine, Palo Alto Veterans Administration Medical Center, Stanford, California 94305, USA.
Mochon
E
E
Arcinas
M
M
Boxer
L M
LM
D000483:Alleles; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D019254:Genes, bcl-2; D006801:Humans; D008223:Lymphoma / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured; D025721:WT1 Proteins
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D025721:WT1 Proteins