The Alternative Breakpoint Region (ABR) of BCL6 Is An Active Transcribed Region in Germinal Center B Cells and May Serve as An Enhancer

Chromosomal translocations at 3q27 involving BCL6 occur in two different regions. Rearrangements with breaks within the major breakpoint region (MBR), comprising the first exon and part of the first intron of BCL6, are among the most common genetic abnormalities in of B-cell non-Hodgkin lymphoma, whereas breaks within an alternative breakpoint region (ABR), located between 245 and 285 kb 5′ to BCL6, have also been reported in follicular lymphoma grade and a small group (6.4%) of diffuse large B-cell lymphomas (DLBCLs). As a result of the MBR translocation, BCL6 expression is deregulated by promoter substitution with either immunoglobulin (Ig) genes or non-Ig genes as partners. A role for deregulated BCL6 expression in the pathogenesis of DLBCL has previously been confirmed in a mouse model. However, the biological role of the more distant ABR region is still not known. Using real-time PCR, we identified in the ABR region an evolutionarily conserved DNase I hypersensitive site (named Far5) which contains a conserved composite binding site for transcription factors PU.1 and IRF4, both of which play important roles in B-cell differentation. Further studies demonstrated that chromatin in the Far5 region, 190kb upstream of BCL6 promoter, has an open configuration in DHL6, Granta 519 and U266 cell lines. Far5 DNA showed enhancer activity by a luciferase reporter assay. PU.1 binds to Far5 in vivo (DHL16 cell line) by a chromatin immunoprecipitation (ChIP) assay, and PU.1 binds in vitro to the conserved PU.1/IRF composite site in Far5 synergistically with either IRF4 or IRF8. ChIP-on-chip assays showed Far5 histone H3K4 monomethylation, a chromatin modification associated with gene enhancers and other regulatory elements. In addition, we identified a transcript upstream of the Far5 region that is specifically expressed in germinal center (GC) B cells, but not at other stages of B-cell differentation. These results indicated that Far5 may play a role in selective expression of intergenic transcripts in GC B-cells. In other genes, intergenic transcription plays a role in looping between distal regulatory regions and the promoter. Our data showed that ABR region is constitutively active in GC B-cells and may play an important role in increasing BCL6 transcription when naïve B cells differentiate into GC B-cells. Further investigation of interactions between the ABR and the BCL6 promoter will uncover the regulatory function of the BCL6 ABR in B-cell differentiation and B-cell lymphomas.