¹Veterans Affairs Palo Alto Health Care System and the Department of Medicine, Center for Molecular Biology in Medicine, Stanford University School of Medicine, Stanford, CA, USA

Correspondence: Dr LM Boxer, Department of Hematology, CCSR 1155, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305-5156, USA. E-mail: lboxer@stanford.edu

Received 11 May 2006; Revised 31 August 2006; Accepted 1 September 2006; Published online 16 October 2006.

Abstract

In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-B (NF-B) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBP bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBP. Reduced expression of C/EBP by transfection of small interfering RNA or interference with NF-B activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.