Integrated Biochemical and Computational Approach Identifies BCL6 Direct Target Genes Controlling Multiple Pathways in Normal Germinal-Center B Cells

BCL6 is a transcriptional repressor required by mature B cells for germinal center (GC) formation, whose deregulated expression, through mutations and translocations, is implicated in lymphomagenesis. The normal function of BCL6 is only partially known since a limited number of direct target genes have been identified by the analysis of cell lines derived from GC-derived lymphomas. However, the complete set of targets functionally relevant for the role of BCL6 in normal GC B cell physiology has not been completely uncovered and the possibility that BCL6 function may be altered in transformed cells has not been excluded. To address these issues, we used an integrated biochemical/computational/functional approach including:

• identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation (ChIP-chip) in normal human GC B cells;

• a computational algorythm inferring transcriptional relationships starting from gene expression data (ARACNe, Basso et al. Nature Genetics 2005);

• validation of physiologic relevance of the candidate target genes by selection of those either not expressed in normal GC B cells or downregulated compared to pre- and post- GC B cells.

This approach identified a set of 1,207 genes which were then subjected to pathway analysis using several databases. The results showed that BCL6 regulates important signaling pathways via direct transcriptional repression of multiple genes acting at different levels from the cell surface (receptors), through signaling molecules to the nucleus. These pathways include:

1. apoptosis, by impairing the expression of both pro- and anti-apoptotic molecules including several TNF-type receptors, signaling molecules (e.g. TRADD, A20, XIAP, TOSO) and effectors (e.g. CASP8);

2. JAK-STAT signaling, through the repression of multiple interleukin and interferon receptors and the transcription factors STAT1, STAT3 and STAT5A;

3. B cell receptor signaling, via repression of signaling molecules (e.g. lyn, vav), several MAP kinases, and transcription factors (e.g. JUN and NF-kB components);

4. Toll-like receptor signaling, by regulating receptors (e.g. TLR1, 7 and 9);

5. DNA damage sensing and response (e.g. TP53BP1, ATM).

Overall, these results provide a comprehensive picture of BCL6 function suggesting that one role of BCL6 is to prevent GC centroblasts from receiving activation and differentiation signals before completion of the phase of proliferative expansion and somatic hypermutation leading to their selection based on antibody affinity maturation.