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 cAMP response element (CRE) in the bcl-2 5'-flanking sequence was identified on the translocated allele. Electrophoretic mobility shift assays with the bcl-2 CRE demonstrated complexes with mobilities identical to those with a consensus CRE. UV cross-linking experiments revealed that proteins with molecular masses of 34, 43, and 67 kDa bound to the bcl-2 CRE site. Electrophoretic mobility shift assay with an antibody specific to the phosphorylated cAMP response-binding protein (CREB) demonstrated that phosphorylated CREB was present in DHL-4 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) led to an increase in both the amount of phosphorylated CREB and the bcl-2 promoter activity. The response to PMA was dependent on an intact CRE site. The activity of the bcl-2 promoter was increased 20-fold in a construct with the immunoglobulin heavy chain enhancers, and mutation of the CRE site abolished most of the induction. The addition of PMA increased the activity of the bcl-2-immunoglobulin enhancer construct by 3.5-fold. Access to the CRE site is blocked in the silent normal bcl-2 allele, while CREB proteins bind to the site on the translocated allele. We conclude that the CRE site functions as a positive regulatory site for the translocated bcl-2 allele in t(14;18) lymphomas.
Highlights
An in Vivo Footprint Is Located over the bcl-2 cAMP-responsive element (CRE) Site—The translocated and normal bcl-2 alleles from DHL-4 cells were separated by pulsed field electrophoresis (Fig. 1)
Nuclear extracts were prepared from DHL-4 cells, and Electrophoretic Mobility Shift Assay (EMSA) was performed with the bcl-2 CRE site and a consensus CRE site
We have used in vivo footprinting to examine the CRE site in the bcl-2 5Ј region, and we have demonstrated that it is occupied on the translocated bcl-2 gene
Summary
IgH, immunoglobulin heavy chain; CRE, c-AMP-responsive element; CREB, CRE-binding protein; ATF, activating transcription factor; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift. CREB Regulates the Translocated bcl-2 Gene gene. 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 IgH enhancers in transient transfection experiments was dependent on the CRE site. Our 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
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