Abstract B14: A biochemical basis for toggling enhancers from the active to poised state in B-cells

The BCL6 transcriptional repressor is required for development of germinal center (GC) B-cells and diffuse large B-cell lymphomas (DLBCL). The BCL6 BTB domain corepressor interaction is an important mediator of BCL6 actions and a potential therapeutic target. BTB point mutations that disrupt corepressor recruitment inactivate BTB domain repressor function. A similar effect can be achieved using specific BCL6 BTB groove binding peptides or small molecules. Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B-cells is unknown. In this study we addressed the question of how BCL6 mediates transcriptional repression in normal and malignant B-cells through its association with SMRT or NCOR corepressor complexes. We combined extensive genomic binding (ChIP-seq) analysis of BCL6, its corepressors SMRT and NCOR and epigenetic chromatin patterns both in DLBCL and in primary human GC B-cells, with functional and biochemical experiments performed in vitro, in cell-based assays and in vivo to reveal the biological and transcriptional mechanism of BCL6. We find that in B-cells, virtually all SMRT/NCOR binding sites overlap with BCL6 binding sites indicating that in these cells BCL6 uniquely sequesters SMRT/NCOR complexes from other factors to mediate cell specific functions. Interestingly, these BCL6-SMRT/NCOR complexes preferentially localize at B cell enhancers. Moreover, BCL6-SMRT/NCOR complexes can inactivate these enhancers by deacetylating H3K27. The SMRT/NCOR-dependent H3K27 de-acetylation is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Furthermore, we show that BCL6 BTB targeted inhibitors may reverse the enhancer silencing effect by disrupting the association of BCL6 with SMRT/NCOR complexes. Taken together these results point to an enhancer “toggling” mechanism that might provide the basis for B-cells to undergo rapid transcriptional changes in response to environmental cues. This mechanism might be particularly relevant to the shuttling of B-cells from the dark to the light zone during affinity maturation facilitating the rapid on/off gene expression changes necessary for this transition. For example genes associated with BCL6-SMRT/NCOR enhancers were highly downregulated in the dark zone compared to the light zone and they were also significantly enriched in genes upregulated by CD40 signaling which occurs in the light zone during B-cell activation by Tfh cells and disrupts the association of BCL6 and SMRT/NCOR. In summary we describe a novel mechanism of enhancer regulation. Reversible enhancer toggling may be critical for dynamic modulation of the BCL6 transcriptional program during the GC reaction as well for the therapeutic effects of BCL6 inhibitors. This mechanism is likely a general biological phenomenon that applies to enhancer regulation in a variety cell types and tumors. Citation Format: Katerina Chatzi, Yanwen Jiang, Chuanxin Huang, Paul Zumbo, Srividya Bhaskara, Alexander D. MacKerell, Jr., Fengtian Xue, Christopher E. Mason, Scott W. Hiebert, Leandro Cerchietti, Vivian J. Bardwell, Olivier Elemento, Melnick Ari. A biochemical basis for toggling enhancers from the active to poised state in B-cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B14.