Jukka Alinikula

Targeting Of Somatic Hypermutation By immunoglobulin Enhancer And Enhancer-Like Sequences

Immunoglobulin gene enhancers have a conserved function in targeting somatic hypermutation to immunoglobulin genes, thereby supporting the production of high affinity antibodies.

Alternate pathways for Bcl6-mediated regulation of B cell to plasma cell differentiation

The transcription factor Bcl6 regulates germinal center formation and differentiation of B cells into high‐affinity antibody‐producing plasma cells. The direct double‐negative regulatory circuit between Bcl6 and Blimp‐1 is well established. We now reveal alternative mechanisms for Bcl6‐mediated regulation of B‐cell differentiation to plasma cells and show with DT40 cells that Bcl6 directly promotes the expression of Bach2, a known suppressor of Blimp‐1. Moreover, Bcl6 suppresses Blimp‐1 expression through direct binding to the IRF4 gene, as well as by promoting the expression of MITF, a known suppressor of IRF4. We also provide evidence that Bcl6 is needed for the expression of AID and UNG, the indispensable proteins for somatic hypermutation and class‐switch recombination, and UNG appears to be a direct Bcl6 target. Our findings reveal a complex regulatory network in which Bcl6 acts as a key element dictating the transition of DT40 B cells to plasma cells.

Ikaros has a crucial role in regulation of B cell receptor signaling

The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cγ2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.

Concerted action of Helios and Ikaros controls the expression of the inositol 5-phosphatase SHIP

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros‐containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR‐signaling strength. In this study, we describe a Helios‐deficient chicken DT40 B‐cell line with a BCR signaling phenotype that is the opposite to that of Ikaros‐deficient cells. In contrast to Ikaros‐deficient cells, Helios−/− B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR‐induced phosphorylation of signaling molecules. The inositol 5‐phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros‐ and Helios‐deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene‐encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

Gene interaction network regulates plasma cell differentiation.

Effective humoral immunity depends on B cells, plasma cells and follicular helper T cells (TFH) and secreted high‐affinity antibodies. The differentiation of mature B cell into plasma cells is ultimately hardwired in a regulatory network of transcription factors. This circuitry is responding to extracellular stimuli, which leads to production of higher‐affinity antibodies after germinal centre (GC) reaction. The understanding of the transcriptional regulation of GCs and the initiation of plasma cell differentiation is becoming increasingly clear. It is evident that transcriptional repressor Blimp‐1 can drive the plasma cell differentiation, but the initiation of plasma cell differentiation in GCs is likely coupled to the loss of B cell characteristics maintained by transcription factors Pax5 and Bcl6.

DT40 mutants: a model to study transcriptional regulation of B cell development and function

A key issue in understanding the hematopoietic system and B cell biology is to define the function of transcription factors. B lymphocyte development and function is controlled by a hierarchy of transcription factors including PU.1, Ikaros, E2A, EBF, Pax5 and Aiolos. Mouse knockout models provide information about the developmental and physiological importance of the disrupted gene. However, an early block in the development or a lethal phenotype prevents the studies of the functional importance of the gene at the later developing system such as the immune system. The chicken B cell line DT40 is used to circumvent these problems. Studies with DT40 have revealed a role for Ikaros transcription factor in B cell receptor signaling and Aiolos has been shown to regulate immunoglobulin gene conversion and cell survival. On the other hand, findings with Pax5 deficient mutants support DT40 targeting system as a valid model for the plasma cell differentiation and demonstrate the genetic plasticity of the cell line. This system is an excellent model to study transcription factors in B cell specific functions, antibody production and B cell differentiation.

A Critical Context-Dependent Role for E Boxes in the Targeting of Somatic Hypermutation

Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following Ag stimulation. In SHM, activation-induced cytidine deaminase mutates the V region of the Ig genes to increase the affinity of Abs. Although SHM acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. In this article, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within “core” targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes are preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, although E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.

Super-Enhancer Transcription Converges on AID

AID mis-targeting is poorly understood but contributes significantly to B cell genome instability. Two new papers in Cell reveal that AID mistargeting occurs primarily in gene bodies within a nuclear microenvironment characterized by high levels of transcriptional activity, interconnected transcriptional regulatory elements, and overlapping sense and antisense (convergent) transcription.

Bach2 regulates AID-mediated immunoglobulin gene conversion and somatic hypermutation in DT40 B cells

The transcription factor Bach2 is required for germinal center formation, somatic hypermutation (SHM), and class‐switch recombination (CSR) of immunoglobulins. SHM and CSR are initiated by activation‐induced cytidine deaminase (AID) which has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID‐mediated immunoglobulin gene diversification processes, we established a BACH2‐deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), another AID‐dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp‐1 and that BACH2‐deficient cells have altered expression of several genes regulating AID expression, stability and function. Furthermore, re‐expression of BACH2 or AID in Bach2KO cells restored the SHM and GCV defects. These results demonstrate that Bach2 has a previously unappreciated role in the production of high‐affinity antibodies.

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

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.