The WT1 Protein Is a Negative Regulator of the Normalbcl-2 Allele in t(14;18) Lymphomas

• Published in: The Journal of biological chemistry 1997-08, Vol.272 (31), p.19609-19614
• Main Authors: Heckman, Caroline, Mochon, Evonne, Arcinas, Magdalena, Boxer, Linda M.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.31.19609

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 vivofootprint 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 thebcl-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 silentbcl-2 allele in t(14;18) lymphomas. The WT1 site is not occupied on the translocated bcl-2 allele.