The BCL6 Oncoprotein Forms Distinct Transcriptional Repression Complexes on Cohorts of Target Genes Involved in Specific Cellular Functions.

The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal B-cells and is frequently constitutively activated in B-cell lymphomas. BCL6 is a transcriptional repressor of the BTB/POZ - zinc finger family of proteins. Transcriptional repressors are generally believed to function by binding to specific DNA sequences in target promoters and then recruiting a cohort of corepressor proteins that physically alter the chromatin structure of the locus leading to silencing. BCL6 can recruit several different corepressors. Accordingly, N-CoR, SMRT and BCoR bind to the BTB domain of BCL6, the NuRD complex binds to the second repression domain and ETO binds to the C-terminal zinc finger domain. These proteins recruit additional corepressors such as HDACS, thus forming multi-protein complexes. BCL6 is involved in both licensing germinal center B-cells for survival as well as blocking differentiation to memory or plasma cells. However, we found that blockade of the BCL6 BTB domain with a specific inhibitor causes only apoptosis but not differentiation of B-cells. In order to identify BCL6 target genes and the mechanism through which they are silenced, we performed extensive ChIP on chip analysis of BCL6, its corepressors, and their chromatin signatures using tiled oligonucleotide arrays containing 1.5 KB of 24,000 promoters. These were performed in the presence or absence of BCL6 shRNA and other BCL6 inhibitor molecules, in tandem with expression arrays as a functional readout. We also performed in depth ChIP on chip experiments using custom arrays, where sets of entire BCL6 target loci were tiled through with overlapping oligos. From these studies we i) identified a large cohort of direct BCL6 target genes involved in apoptosis, cell damage, protein degradation and differentiation that provide critical insight into the mechanism of action of BCL6 in normal and malignant B-cells; ii) discovered that cohorts of BCL6 direct target genes involved in different pathways are regulated by specific corepressors, which are mutually exclusive in their binding to BCL6 target loci, iii) that BCL6 can repress genes through a variety of different mechanisms with unique chromatin signatures. Thus, previously unrecognized mechanisms exist in transcriptional repression, that determine formation of specific corepressor complexes. Using this information, we have been able to re-activate discrete cohorts of BCL6 target genes controlled by specific corepressors, resulting in specific biological effects such as apoptosis or differentiation of lymphoma cells. These results provide fundamental insights into the transcriptional and biological mechanism of action of BCL6 in B-cells.