Identification of negative regulatory regions within the first exon and intron of the BCL6 gene

Abstract

Chromosomal translocations involving BCL6 gene are frequent in human B-cell lymphomas. Chromosomal breaks preferentially occur within a 3-kb region containing the ®rst exon and intron. Recent reports have revealed that internal deletions or point mutations also are common in this region, suggesting that structural alteration of this region may be a crucial event in the development of lymphomas. In this study, we identi®ed two regions in the BCL6 gene that negatively regulate BCL6 expression. One region, ES, is located within the ®rst exon between nucleotides +472 and +543, and a second region, IS, is located between +783 and +918 of the ®rst intron. A consensus nucleotide sequence for the binding of the BCL6 protein itself was found within the ES region. An electrophoretic mobility shift assay and a co-transfection experiment using a BCL6 expression vector showed that transcription of the BCL6 gene was negatively regulated by the BCL6 gene product. The IS region which is included in the regions commonly deleted in B-cell lymphomas had a silencer activity. Structural alterations of these two regions may play roles in the deregulated expression of the BCL6 gene in B-cell lymphomas. Oncogene (2000) 19, 4941 ± 4945.Keywords: BCL6; B-cell lymphoma; silencer element A chromosomal translocation involving 3q27 is one of the most common cytogenetic abnormalities in human B-cell lymphomas. The BCL6 gene, encoding a zinc®nger transcriptional repressor, has been isolated from the breakpoint at 3q27 (Baron et al., 1993;Kerckaert et al., 1993;Miki et al., 1994;Ye et al., 1993). The translocation preferentially occurs within a 3-kb region spanning the ®rst exon-intron boundary. The promoter region and ®rst non-coding exon are replaced by other genes including the immunoglobulin (Ig) genes, and the resulting deregulated expression of BCL6 may contribute to lymphomagenesis.Recent studies have found that some lymphomas carry internal deletions (Bernardin et al., 1997;Nakamura et al., 1996;1999), and more frequently, point mutations Gaidano et al., 1997;Liang et al., 1998;Migliazza et al., 1995) within the BCL6 gene. Interestingly, the deletions and mutations preferentially occur within the ®rst exonintron boundary region, the same region in which the breakpoints for chromosomal translocation cluster. These observations suggest that the structural alteration of this region, which may contain regulatory sequences, is a crucial event in the development of Bcell lymphomas.To investigate the functional signi®cance of the ®rst exon-intron boundary region on the transcriptional regulation of the BCL6 gene, luciferase reporter constructs carrying various genomic fragments of this region were transfected transiently into Raji cells, expressing endogenous BCL6 ( Figure 1A). pBCL7657/471Luc, containing the region from 7657 to +471, showed signi®cant luciferase activity in Raji cells, as previously reported (Ohashi et al., 1995). We arbitrarily de®ned the luciferase activity of pBCL7657/471Luc as 100% and used this as a reference to normali...

IgV and bcl6 somatic mutation analysis reveals the heterogeneity of cutaneous B-cell lymphoma, and indicates the presence of undisclosed local antigens

“…33,34 Bcl6 somatic mutations are described in germinal center (GC) and post-GC-derived lymphoma, probably caused by somatic mutation machinery acting on the 5 0 noncoding region of the bcl6 gene, due to its sequence homology with immunoglobulin gene. 27,35,36 Our analysis showed mutations of bcl6 5' noncoding region in two of three pCFLs, and one of two pDBCLs and sDBCL. These mutational findings were observed in previous reports of nodal lymphomas, where no differences in bcl6 mutation rate in FL and DBCL have been observed.…”

STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas

“…These mutations may dramatically change the regulation of BCL6 expression, resulting in overexpression of BCL6. One possible mechanism may be through loss of autoregulation by BCL6 itself (Kikuchi et al, 2000;Pasqualucci et al, 2003). Another mechanism may be through loss of binding of other negative regulatory elements.…”

The BCL6 gene in B-cell lymphomas with 3q27 translocations is expressed mainly from the rearranged allele irrespective of the partner gene

“…The first exon region of the BCL-6 gene, which has previously been shown to contain BCL6 binding regulatory sequences, 29,30 was amplified by PCR using 5 0 -ACGCTCTGCTTATGAGGA and 5 0 -CGGCAGCAACAGCAATAA primers. The PCR amplicons were sequenced on a 373 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) using ABI Prism Big Dye Terminator Kit (Perkin Elmer, USA) as recommended by the manufacturer.…”