THE WT1 PROTEIN IS A NEGATIVE REGULATOR OF THE NORMAL BCL-2 ALLELE INT(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 electrophore sis. An in vivo footprint over a potential WT1 binding site in the bcl -2 5'-flanking sequence was identified on the normal silent allele. El ectrophoretic mobility shift assays with the bcl-2 WT1 site demonstrat ed a single specific complex. UV cross-linking and Western analysis re vealed that this gel shift complex contained WT1 protein. Deletion or mutation of the WT1 site resulted in an increase in activity of the bc l-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 re pression of the bcl-2 promoter. Cotransfection with a WT1 expression v ector and the bcl-2 promoter with the mutated WT1 site resulted in onl y 1.2-fold repression. We conclude that the WT1 site functions as a ne gative 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 alle le.