CREB proteins function as positive regulators of the translocated bcl-2 allele in t(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 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.
Ji
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Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center, Stanford, California 94305, USA.
Mochon
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Arcinas
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Boxer
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D000483:Alleles; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D018983:DNA Footprinting; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D008223:Lymphoma / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014178:Translocation, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D007143:Immunoglobulin Heavy Chains; D019253:Proto-Oncogene Proteins c-bcl-2; D013755:Tetradecanoylphorbol Acetate