Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

Abstract

The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

Transcriptomic analysis of early B-cell development in the chicken embryo

“…Additional experiments showed that Bcl6 transactivates Bach2 and targets the AICDA gene in cooperation with Pax5 (Alinikula et al, 2011). While Bcl6 not only transactivates AICDA, the activation of additional genes by Bcl6 are also required for Ig–gene conversion (Williams et al, 2016). Moreover, a Bach2 deficiency in the same cell line abrogated Ig–gene conversion and showed that Bach2 may transactivate the AICDA promoter as well as transactivate genes associated with function of the AID protein (Budzynska et al, 2017).…”

Immunoglobulin enhancers increase RNA polymerase 2 stalling at somatic hypermutation target sequences

“…The ChIPs were done essentially as previously described (Williams et al, 2016), with the following modifications. 4 × 10 7 formaldehyde-fixed cells were lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, supplemented with Complete, EDTA-free Protease Inhibitor Cocktail (Roche)) and diluted 4-fold with dilution buffer (16.4 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, supplemented with inhibitors).…”

BLIMP‐1 is insufficient to induce antibody secretion in the absence of IRF4 in DT40 cells

“…We reasoned that immortalized DT40 cells overexpressing MYC would allow to more precisely dissect the roles of IRF4 and BLIMP‐1 in antibody secretion. This cell line is derived from chicken bursal follicles, where BCL6 and BACH2 are required for somatic hypermutation and gene conversion . We have previously shown that deletion of PAX5 or BCL6 in these cells leads to spontaneous upregulation of PRDM1 and differentiation into early ASC phenotype …”

“…This cell line is derived from chicken bursal follicles, where BCL6 and BACH2 are required for somatic hypermutation and gene conversion. 14,15 We have previously shown that deletion of PAX5 or BCL6 in these cells leads to spontaneous upregulation of PRDM1 and differentiation into early ASC phenotype. 4,16 In this study, we tested whether IRF4 is needed for antibody secretion and PRDM1 expression.…”