Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells

Abstract

BCL6 is a transcriptional repressor required for mature B-cell germinal center (GC) formation and implicated in lymphomagenesis. BCL6's physiologic function is only partially known because the complete set of its targets in GC B cells has not been identified. To address this issue, we used an integrated biochemical-computational-functional approach to identify BCL6 direct targets in normal GC B cells. This approach includes (1) identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation, (2) inference of transcriptional relationships by the use of a regulatory network reverse engineering approach (ARACNe), and (3) validation of physiologic relevance of the candidate targets down-regulated in GC B cells. Our approach demonstrated that a large set of promoters (> 4000) is physically bound by BCL6 but that only a fraction of them is repressed in GC B cells. This set of 1207 targets identifies several cellular functions directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine signaling, Toll-like receptor signaling, and differentiation. These results define a broad role of BCL6 in preventing centroblasts from responding to signals leading to exit from the GC before they complete the phase of proliferative expansion and of antibody affinity maturation.

Figure 1

Motifs associated with BCL6 binding and their distribution across promoters. (A) DNA sequence analysis of BCL6 bound promoters as detected by ChIP-on-chip revealed enrichment for 3 motifs (M00424, M2, and M0), of which 1 (M0) resembles previously reported BCL6 binding sequences (B6BS, BCL6). (B) The module including M0, M00424, and M2 within 375 bp is found in the majority of BCL6-bound promoters, the M0 motif has sites in the promoters of the majority of the remaining genes, and almost half of the M0-free promoters display Inr elements. BCL6 is known to repress some of its targets upon interaction with Miz-1, which binds to Inr elements.

Figure 2

The M0 site is necessary and sufficient for BCL6 repression on the PGM2L1 promoter. (A) Schematic representation of BCL6 binding to the PGM2L1 promoter as detected by ChIP-on-chip and of luciferase reporter constructs generated to assess the role of M0, M2, and M00424 sites. (B) BCL6 binding to PGM2L1 promoter was confirmed by qChIP assay in GC B cells obtained from 2 donors. A neighboring region (PGM2L1-CTR; −2351/−2546) tested as a control was not enriched. (C) A luciferase reporter construct driven by the PGM2L1 promoter was cotransfected in 293T cells with plasmids expressing HA-BCL6 or its mutants lacking the DNA binding domain (HA-BCL6ΔZF) or the transcriptional repressor domain (HA-BCL6 ZF). The PGM2L1 promoter-driven luciferase construct showed a dose-dependent decreased luciferase activity in the presence of HA-BCL6 but not of its mutants. The relative luciferase activities are displayed as average ± SD of 2 independent transfections. Immunoblotting protein detection of HA-BCL6 wild-type and mutants is displayed on the right. Results are representative of at least 3 independent experiments each performed in duplicate. (D) Disruption of M00424 and M2 sites in PGM2L1 promoter (mutant 1) did not affect repression by BCL6. Conversely, mutations in M0 (mutant 2) were linked to resistance to BCL6 repression, and further mutagenesis of all sites (mutant 3) did not increase the level of luciferase activities. The relative luciferase activities are displayed as average ± SD of 2 independent transfections. Results are representative of at least 3 independent experiments, each performed in duplicate.

Figure 3

BCL6 modulates multiple pathways in normal GC B cells. BCL6 represses multiple genes at different levels from the membrane surface (receptors), through signal transduction molecules, into the nucleus (transcription factors). Representative genes and gene families, found to be targeted by BCL6, are displayed for each pathway. Details on BCL6 targets and their pathways are reported in supplemental Figure 7. Several pathways (BCR and CD40 signaling; DNA damage response) have been previously reported to have a repressor function on BCL6, leading to its transcriptional down-regulation and protein degradation.

Figure 4

BCL6 targets involved in multiple pathways are responsive to BCL6 silencing. (A) Seventeen targets representative of the major pathways affected by BCL6 were analyzed in the P3HR1 cell line. The BCL6-bound promoter region identified by ChIP-on-chip in GC B cells was tested for direct binding in P3HR1 by qChIP. All promoters, except JAK3, showed enrichment of at least 2-fold in the BCL6 immunoprecipitated chromatin fraction. (B) BCL6 silencing was obtained by the use of 2 shRNAs delivered by lentiviral infection in P3HR1. Quantitative reverse-transcription PCR analysis showed that the expression of 11 of 17 targets increased more than 2-fold upon BCL6 silencing by at least one of the shRNA. The target induction appeared consistent with the level of BCL6 silencing in all cases, except STAT5A. (C) BCL6 protein expression as detected by immunoblotting upon infection with control and BCL6 shRNAs.

Figure 5

BCL6-silencing gene signature is significantly enriched in BCL6 target genes. Gene set enrichment according to up-regulation in response to BCL6 silencing was measured for (A) the targets identified as BCL6-anti-coexpressed genes with BCL6-bound promoters according to ChIP-on-chip analysis and (B) the subset of targets that is also inferred by ARACNe. GSEA computed the significance of up-regulation in BCL6-silencing versus control experiments by t test (top meter in each panel) for each target set (second meter). Leading edge analysis was used to predict responsive BCL6 targets (left of vertical line in each panel) and enrichment score (ES) P values were estimated by the use of extrapolation over 100 000 sample-permutation tests.