Abstract
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.
Highlights
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
We identified an in vivo footprint at a cAMP-responsive element (CRE) site in the 59-flanking sequence of the translocated bcl-2 gene, and we demonstrated that CREB family proteins bound to this site in vitro and that the maximal increase in bcl-2 promoter expression mediated by the immunoglobulin heavy chain gene enhancers in transient transfection experiments was dependent on the CRE site [11]
We describe an in vivo footprint over a potential WT1 site on the normal bcl-2 allele; this site is not occupied on the translocated allele
Summary
Vol 272, No 31, Issue of August 1, pp. 19609 –19614, 1997 Printed in U.S.A. The WT1 Protein Is a Negative Regulator of the Normal bcl-2 Allele in t(14;18) Lymphomas*. We identified an in vivo footprint at a CRE site in the 59-flanking sequence of the translocated bcl-2 gene, and we demonstrated that CREB family proteins bound to this site in vitro and that the maximal increase in bcl-2 promoter expression mediated by the immunoglobulin heavy chain gene enhancers in transient transfection experiments was dependent on the CRE site [11]. These results suggest that the CRE site functions as a positive regulatory element for the translocated bcl-2 allele in follicular lymphoma with the t(14; 18) translocation. The presence of the immunoglobulin 39-enhancers abrogates the repression by WT1
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