Reversible disruption of BCL6 repression complexes by CD40 signaling in normal and malignant B cells

Abstract

Germinal center (GC) B cells undergo somatic hypermutation, class switch recombination, and rapid clonal expansion to produce high-affinity antibodies. The BCL6 transcriptional repressor facilitates this phenotype because it can repress DNA damage checkpoint genes. GC B and T cells can make transient direct physical contact; T cells were observed to be associated with dead B-cell fragments. We thus hypothesized that one function of CD40 signaling from T cells within this timeframe could be to modulate BCL6 activity. CD40 signaling rapidly disrupts the ability of BCL6 to recruit the SMRT corepressor complex by excluding it from the nucleus, leading to histone acetylation, RNA polymerase II processivity, and activation of BCL6 target genes, such as CD23b, ATR, and TP53. Washout of CD40 to emulate transient T-cell contact permitted BCL6 target gene mRNA levels to return to their repressed levels, demonstrating that this is a reversible process, which could allow centroblasts that pass quality control to either continue proliferation or undergo terminal differentiation. These data suggest that transient CD40 signaling in the GC might allow T cells to weed out heavily damaged centroblasts while at the same time promoting survival of intact B cells, which could undergo differentiation or additional rounds of proliferation.

Figure 1

CD40 signaling rapidly up-regulates CD23b expression before down-regulation of BCL6. (A) Ramos cells were exposed to CD40L for the indicated durations, after which QPCR was performed to detect the mRNA abundance of the CD23b gene product. CD23b mRNA levels were first normalized by GAPDH levels and expressed as fold change compared with untreated cells. (B) BCL6 mRNA levels were detected by QPCR in the same cells as in panel A. Panels A and B correspond to the average of 3 independent replicates. (C) Western blots were performed in cells treated as in panels A and B using antibodies for BCL6 and for actin as a loading control. (D) Densitometry of 3 independent BCL6 Western blot replicates. The signal of each BCL6 Western blot was first normalized by actin levels and then expressed in fold compared with untreated cells. Error bars represent standard error of the mean (SEM).

Figure 2

CD40 rapidly ejects corepressors from the BCL6 repression complex on the CD23b promoter. (A) Chromatin immunoprecipitations were performed in Ramos cells at the indicated time points of CD40L exposure using BCL6 antibody. QPCR was performed to measure the enrichment of the CD23b promoter BCL6 binding site compared with input chromatin. The BCL6 enrichment values were first normalized to the input levels and expressed relative to untreated cells. The results correspond to the average of 3 independent replicates. (B) Chromatin immunoprecipitations were performed with SMRT and HDAC3 antibodies at the indicated time points after CD40L exposure to Ramos cells. The enrichment values for the different corepressors were first normalized to input levels and expressed relative to untreated cells. The results correspond to the average of 3 independent replicates. Error bars represent SEM.

Figure 3

CD40L triggers posttranslation modification of SMRT and its translocation to the cytoplasm. (A) Western blots were performed using SMRT antibody in Ramos cells exposed to CD40L during the indicated time points. Actin Western blots were performed as a loading control. As previously reported, SMRT is represented by 2 bands, a faster migrating product (SMRT) and a slower band (SMRT-X) indicative of a posttranslational modification. (B) The densitometry of 3 independent SMRT Western blot replicates. The signal of each SMRT Western blot was first normalized by actin levels and then expressed as fold change relative to untreated cells. ■ represents the faster migrating band; , the slower migrating posttranslationally modified form of SMRT. Error bars represent SEM. (C) Ramos cells were transfected with a GFP-SMRT-expressing plasmid and then exposed to CD40L for 2 hours. Immunofluorescence and phase contrast microscopy were performed to determine the cellular localization of transfected SMRT. Rows 3 to 5 show experiments in which transfected Ramos cells were pretreated with the MEK kinase inhibitors PD98059 and UO126 or vehicle, followed by exposure to CD40L.

Figure 4

The chromatin of the CD23b locus is set to a transcriptionally active state by CD40 signaling, leading to recruitment and processivity of POLII. (A) QChIP assays were performed using anti-pan-Histone 3 acetylated, anti-pan-Histone 4 acetylated and antiactin antibodies in Ramos cells treated with CD40L during 0 (□), 30 minutes (), and 2 hours (■). QPCR was performed to detect the abundance of CD23b promoter sequence as in Figure 2. The y-axis represents the percentage enrichment by each antibody relative to input. (B) QChIP was performed at the indicated time points using RNA POLII antibodies to detect the abundance of POLII at the CD23b promoter, exon1 and exon 4. The y-axis represents the percentage enrichment of the amplicons relative to input. The result shows increase occupancy by POLII at exon 1 and exon 4 after CD40L exposure, consistent with active transcription of the gene. Each experiment was carried out in duplicate. Error bars represent SEM.

Figure 5

CD40 signaling induces several BCL6 target genes in lymphoma cells and primary centroblasts. (A) Ramos cells were exposed to CD40L for 0, 0.5, 2, 8, or 24 hours, after which QPCR was performed to detect the mRNA abundance of BCL6, ATR, p53, and CCL3. The mRNA levels were first normalized by GAPDH levels and expressed as fold change relative to untreated cells. (B) Purified CD77⁺ centroblasts were exposed to CD40L for 0, 0.5, 2, 4, 8, or 24 hours after which QPCR was performed to detect the mRNA abundance of BCL6, ATR, CCL3, and CD23b. The mRNA levels were first normalized by GAPDH levels and shown as fold change relative to untreated cells. These experiments were performed in triplicate. Error bars represent SD.

Figure 6

CD40-mediated up-regulation of BCL6 target genes is reversible. Ramos cells were exposed to CD40L for 1 hour and then split into 3 fractions. The first fraction was immediately lysed (), the second was washed to remove the CD40L and then cultured for an additional 5 hours (▨), and the third fraction remained in CD40L containing media for another 5 hours (■). QPCR was performed in untreated cells and all 3 fractions to detect mRNA abundance of BCL6, ATR, CCL3, and CD23b. The mRNA levels of these genes were first normalized to GAPDH mRNA levels and expressed as fold increase relative to untreated cells. The experiment was performed in triplicate. Error bars represent SEM.

Figure 7

CD40 signaling can block the function of BCL6 through 2 independent mechanisms. (A) In dark zone centroblasts, BCL6 represses target genes through recruitment of the SMRT and N-CoR corepressors, both of which form histone deacetylase (HDAC) complexes. This facilitates proliferation and immunoglobulin affinity maturation. (B) CD40 signaling in the light zone, for example, by GC T cells, leads to posttranslational modification of these corepressors () and loss of their association with BCL6. This leads to a failure to maintain silencing of these genes, which can now become reactivated even in the presence of BCL6. The double arrow between dark zone and light zone indicates that this is a reversible mechanism. This mechanism is rapid and may allow damaged B cells to be removed from the GC reaction. (C) In the second mechanism, sustained CD40 signaling, for example, by follicular dendritic cells, can activate NFκB, which in turn induces expression of IRF4, which can then directly repress BCL6 mRNA expression leading to down-regulation of BCL6 and up-regulation of its target genes. This mechanism is slower but is irreversible and leads to differentiation of B cells positively selected for high-affinity antibody.