Volume 272, Number 31, Issue of August 1, 1997 pp. 19609-19614
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, March 17, 1997, and in revised form, May 23, 1997)

From the Center for Molecular Biology in Medicine, Palo Alto Veterans Administration Medical Center and the Department of Medicine, Stanford University School of Medicine, Stanford, California 94305

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.

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