PI-1 BINDING-SITES ARE NEGATIVE REGULATORS OF BCL-2 EXPRESSION IN PRE-B CELLS

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression i n mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identi fied two negative regulatory regions in the human bcl-2 5' flanking an d 5' untranslated regions in pre-B cells; these regions have no signif icant function in mature B cells. Further investigation of these regio ns revealed two pre-B cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulator y region. Mutational analysis confirmed that these three sites functio ned as negative regulators of the bcl-2 promoter in the pre-B-cell lin e Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed wit h the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bo und to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins, Con structs with the isolated pi 1 sites linked to the simian virus 40 pro moter were used in transient transfection experiments in the pre-B cel l line. The bcl-2 sites decreased expression of the simian virus 40 pr omoter, while the immunoglobulin enhancer site increased its expressio n. The pi sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.