IgH Transcription Research Articles

Comprehensive Multi-Omics Analysis of Gene Fusions in a Large Multiple Myeloma Cohort

Introduction: Gene fusions are the result of genomic rearrangements that create hybrid protein products or bring the regulatory elements of one gene into close proximity of another. Fusions often dysregulate gene function or expression through oncogene overexpression or tumor suppressor underexpression (Gao, Liang, Foltz, et al. Cell Rep 2018). Some fusions such as EML4–ALK in lung adenocarcinoma are known druggable targets. Fusion detection algorithms utilize discordantly mapped RNA-seq reads. Careful consideration of detection and filtering procedures is vital for large-scale fusion detection because current methods are prone to reporting false positives and show poor concordance.Multiple myeloma (MM) is a blood cancer in which rapidly expanding clones of plasma cells spread in the bone marrow. Translocations that juxtapose the highly-expressed IGH enhancer with potential oncogenes are associated with overexpression of partner genes, although they may not lead to a detectable gene fusion in RNA-seq data. Previous studies have explored the fusion landscape of multiple myeloma cohorts (Cleynen, et al. Nat Comm 2017; Nasser, et al. Blood 2017). In this study, we developed a novel gene fusion detection pipeline and post-processing strategy to analyze 742 patient samples at the primary time point and 64 samples at follow-up time points (806 total samples) from the Multiple Myeloma Research Foundation (MMRF) CoMMpass Study using RNA-seq, WGS, and clinical data.Methods and Results: We overlapped five fusion detection algorithms (EricScript, FusionCatcher, INTEGRATE, PRADA, and STAR-Fusion) to report fusion events. Our filtered call set consisted of 2,817 fusions with a median of 3 fusions per sample (mean 3.8), similar to glioblastoma, breast, ovarian, and prostate cancers in TCGA. Major recurrent fusions involving immunoglobulin genes included IGH–WHSC1 (88 primary samples), IGL–BMI1 (29), and the upstream neighbor of MYC, PVT1, paired with IGH (6), IGK (3), and IGL (11).For each event, we used WGS data when available to determine if there was genomic support of the gene fusion (based on discordant WGS reads, SV event detection, and MMRF CoMMpass Seq-FISH WGS results) (Miller, et al. Blood 2016). WGS validation rates varied by the level of RNA-seq evidence supporting each fusion, with an overall rate of 24.1%, which is comparable to previously observed pan-cancer validation rates using low-pass WGS.We calculated the association between fusion status and gene expression and identified genes such as BCL2L11, CCND1/2, LTBR, and TXNDC5 that showed significant overexpression (t-test). We explored the clinical connections of fusion events through survival analysis and clinical data correlations, and by mining potentially druggable targets from our Database of Evidence for Precision Oncology (dinglab.wustl.edu/depo) (Sun, Mashl, Sengupta, et al. Bioinformatics 2018). Major examples of upregulated fusion kinases that could potentially be targeted with off-label drug use include FGFR3 and NTRK1.We examined the evolution of fusion events over multiple time points. In one MMRF patient with a t(8;14) translocation joining the IGH locus and transcription factor MAFA, we observed IGH fusions with TOP1MT (neighbor of MAFA) at all four time points with corresponding high expression of TOP1MT and MAFA. Using non-MMRF single-cell RNA data from different patients, we were able to track cell-type composition over time as well as detect subpopulations of cells harboring fusions at different time points with potential treatment implications.Discussion: Gene fusions offer potential targets for alternative MM therapies. Careful implementation of gene fusion detection algorithms and post-processing are essential in large cohort studies to reduce false positives and enrich results for clinically relevant information. Clinical fusion detection from untargeted RNA-seq remains a challenge due to poor sensitivity, specificity, and usability. By combining MMRF CoMMpass data from multiple platforms, we have produced a comprehensive fusion profile of 742 MM patients. We have shown novel gene fusion associations with gene expression and clinical data, and we identified candidates for druggability studies. DisclosuresVij:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Energetics and Physiological Impact of Cohesin Extrusion

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized invivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA.We also identify architectural "stripes," where a loop anchor interacts with entire domains at highfrequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region.

The IgH 3' regulatory region (3'RR) controls class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The mouse 3'RR contains four enhancer elements with hs1,2 flanked by inverted repeated sequences and the centre of a 25-kb palindrome bounded by two hs3 enhancer inverted copies (hs3a and hs3b). hs4 lies downstream of the palindrome. In mammals, evolution maintained this unique palindromic arrangement, suggesting that it is functionally significant. Here we report that deconstructing the palindromic IgH 3'RR strongly affects its function even when enhancers are preserved. CSR and IgH transcription appear to be poorly dependent on the 3'RR architecture and it is more or less preserved, provided 3'RR enhancers are present. By contrast, a ‘palindromic effect' significantly lowers VH germline transcription, AID recruitment and SHM. In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance.

The IgH 3′ regulatory region influences lymphomagenesis in Igλ-Myc mice

The IgH 3'regulatory region (3'RR), encompassing the four transcriptional enhancers hs3a-hs1,2-hs3b-hs4, has a key role on class switch recombination, somatic hypermutation, IgH transcription and B-cell fate. In plasma cells, transcribed IgH and IgL loci often colocalized in transcription factories and an IgL transcription defect might translate into lowered IgH transcription. We explored whether the 3'RR would affect lymphomagenesis in Igλ-Myc transgenic mice prone to lymphoproliferations. Breeding Igλ-Myc transgenics in a background deficient for the 3'RR influences lymphomagenesis toward less mature lymphomas (16% vs 54%, p = 0.01, Z test for two population proportions). In a 3'RR-deficient background mature tumors less often expressed the CD43 antigen (54% vs 0%, p = 0.02), a membrane glycoprotein expressed on activated mature B-cells. In contrast, in a 3'RR-deficient background tumors more often expressed the CD5 antigen (32% vs 12%, p = 0.05) that may serve to control autoimmunity and that is suspected to play a role in leukemic transformation. Lymphoma myc transcript levels, the Ki67 index of proliferation, the clonality, the usage of V(D)J segments, and their somatic hypermutation status were not affected in the 3'RR-deficient background. In conclusion, most probably through its action during the maturation process, the 3'RR can influence lymphomagenesis even when not linked with an oncogene.

3′RR targeting in lymphomagenesis: a promising strategy?

3′RR targeting in lymphomagenesis: a promising strategy?

The aryl hydrocarbon receptor regulates an essential transcriptional element in the immunoglobulin heavy chain gene

Ig heavy chain (Igh) transcription involves several regulatory elements including the 3′Igh regulatory region (3′IghRR). 3′IghRR activity is modulated by several transcription factors, including NF-κB and AP-1 and potentially the aryl hydrocarbon receptor (AhR). The prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits antibody secretion and 3′IghRR activity. However, the exact mechanism is unknown and TCDD can modulate NF-κB and AP-1 in an AhR-independent manner. To determine if the AhR is a significant regulator of the 3′IghRR, we utilized a mouse B-cell line that stably expresses a 3′IghRR-regulated transgene and either an AhR antagonist or shRNA targeting the AhR. Disruption of the AhR pathway reversed TCDD-induced suppression of the 3′IghRR-regulated transgene and of endogenous Ig demonstrating a biologically significant effect of the AhR on 3′IghRR activation. Altered human 3′IGHRR activity by AhR ligands, which include dietary, environmental, and pharmaceutical chemicals, may have significant implications to human diseases previously associated with the 3′IGHRR.

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: Modulation by BCR and CD40, and relevance to T-independent antibody responses

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.

The B-Cell Receptor Is Required for Optimal Viability, Growth, and Chemotherapy Resistance of Diffuse Large B-Cell Lymphoma Cell Lines of the Germinal Center B-Cell Subtype

Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL.Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting.Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells.GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO.Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways.TableA.B.Figure 1. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of a new ARID family transcription factor (Brightlike/ARID3C) that co-activates Bright/ARID3A-mediated immunoglobulin gene transcription

Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eμ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright.

Interaction between the immunoglobulin heavy chain 3′ regulatory region and the IgH transcription unit during B cell differentiation

The immunoglobulin heavy (Igh) chain locus is subject to precisely regulated processes, such as variable region gene formation through recombination of variable (VH), diversity (DH), and joining (JH) segments, class switching and somatic hypermutation. The 3′ regulatory region (3′ RR) is a key regulator of the Igh locus, and, as revealed by deletions in mouse plasma cell lines and mice, is required for IgH expression as well as class switching. One of the mechanisms by which the 3′ RR regulates its targets is through long-range physical interactions. Such interactions between elements of the 3′ RR and a target site in the IgH transcription unit have been detected in plasma cells, and in resting and switching B cells, where they have been associated with IgH expression and class switching, respectively. Here, we report that lentiviral shRNA knockdown of transcription factors, CTCF, Oct-2, or OBF-1/OCA-B, had no discernible defects in loop formation or H chain expression in plasma cells. JH–3′ RR interactions in pre-B cell lines were specifically associated with IgH expression. JH–3′ RR interactions were not detected in either Pax5-deficient or RAG-deficient pro-B cells, but were apparent in an Abelson-derived pro-B cell line. These observations imply that the 3′ RR has different loop interactions with target Igh sequences at different stages of B cell development and Igh regulation.

Hydrogen peroxide modulates immunoglobulin expression by targeting the 3′Igh regulatory region through an NFκB-dependent mechanism

Reactive oxygen species such as hydrogen peroxide (H2O2) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H2O2 on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3′Igh regulatory region (3′IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H2O2 can enhance 3′IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H2O2 or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H2O2 concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H2O2 can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3′IghRR activity. These results may have significant implications to disease states previously associated with the 3′IghRR.

Direct Interactions of OCA-B and TFII-I Regulate Immunoglobulin Heavy-Chain Gene Transcription by Facilitating Enhancer-Promoter Communication

B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in Igh promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated Igh promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3' enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3' enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism.

Comparison of Identical and Functional Igh Alleles Reveals a Nonessential Role for Eμ in Somatic Hypermutation and Class-Switch Recombination

Somatic hypermutation (SHM), coupled with Ag selection, provides a mechanism for generating Abs with high affinity for invading pathogens. Class-switch recombination (CSR) ensures that these Abs attain pathogen-appropriate effector functions. Although the enzyme critical to both processes, activation-induced cytidine deaminase, has been identified, it remains unclear which cis-elements within the Ig loci are responsible for recruiting activation-induced cytidine deaminase and promoting its activity. Studies showed that Ig gene-transcription levels are positively correlated with the frequency of SHM and CSR, making the intronic, transcriptional enhancer Eμ a likely contributor to both processes. Tests of this hypothesis yielded mixed results arising, in part, from the difficulty in studying B cell function in mice devoid of Eμ. In Eμ's absence, V(H) gene assembly is dramatically impaired, arresting B cell development. The current study circumvented this problem by modifying the murine Igh locus through simultaneous insertion of a fully assembled V(H) gene and deletion of Eμ. The behavior of this allele was compared with that of a matched allele carrying the same V(H) gene but with Eμ intact. Although IgH transcription was as great or greater on the Eμ-deficient allele, CSR and SHM were consistently, but modestly, reduced relative to the allele in which Eμ remained intact. We conclude that Eμ contributes to, but is not essential for, these complex processes and that its contribution is not as a transcriptional enhancer but, rather, is at the level of recruitment and/or activation of the SHM/CSR machinery.

Genomic deletion of the whole IgH 3′ regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes

AbstractThe immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation. In progenitor B cells, V(D)J assembly allows expression of µ heavy chains. In mature B cells, class switch recombination may replace the expressed constant (C)µ gene with a downstream CH gene. Finally, plasma cell differentiation strongly boosts IgH transcription. How the multiple IgH transcriptional enhancers tune these changes is unclear. Here we demonstrate that deletion of the whole IgH 3′ regulatory region (3′RR) allows normal maturation until the stage of IgM/IgD expressing lymphocytes, but nearly abrogates class switch recombination to all CH genes. Although plasma cell numbers are unaffected, we reveal the role of the 3′RR into the transcriptional burst normally associated with plasma cell differentiation. Our study shows that transcriptional changes and recombinations occurring after antigen-encounter appear mainly controlled by the 3′RR working as a single functional unit.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Immunoglobulin Heavy Chain Exclusion in the Shark

The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig) in B lymphocytes and T cell receptors (TCR) in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H) chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15–200 independently rearranging miniloci (VH-D1-D2-JH-Cμ), a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell) examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus.

IgH intronic enhancer, Eμ, and associated matrix attachment regions are required for efficient somatic hypermutation (35.1)

Abstract Somatic hypermutation is a critical mechanism for generating antibody with high affinity. Although the enzyme critical to this process, AID, has been identified, the mechanism that largely limits its activity to the Ig loci is not known. Prior studies have shown that IgH gene transcription levels are positively correlated with somatic hypermutation frequency. The intronic enhancer, Eμ, both stimulates IgH transcription and is essential for promoting efficient V region gene assembly in the IgH locus. It consists of both a core region (cEμ) and two flanking matrix attachment regions (MARs). A recent study reported normal levels of somatic hypermutation in the B cells of cEμ-deficient mice. In the present study, we tested whether deletion of both cEμ and MARs had an effect on somatic hypermutation. We found that the mutation frequency for the Eμ-deficient allele was reduced to approximately 1/3 of wild-type levels. This dramatic decrease in mutation frequency suggests that cEμ, together with its associated MARs, plays an important role in somatic hypermutation within the IgH locus. Supported by NIH grant AI030653 to L.A.E. Research infrastructure and instrumentation at Hunter College is supported by NIH RCMI grant RR-03037.

A Novel ARID/Bright-like Protein Involved in Transcriptional Activation of Cyst Wall Protein 1 Gene in Giardia lamblia

The capability of protozoan parasite Giardia lamblia to encyst is critical for survival outside the host and its transmission. AT-rich interaction domain (ARID) or Bright homologs constitute a large family of transcription factors in higher eukaryotes that regulate cell proliferation, development, and differentiation. We asked whether Giardia has ARID-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome data base identified two genes with putative ARID/Bright domains (gARID1 and 2). Epitope-tagged gARID1 was found to localize to nuclei. Recombinant gARID1 specifically bound to the encystation-induced cyst wall protein (cwp) gene promoters. Mutation analysis revealed that AT-rich initiators were required for binding of gARID1 to the cwp promoters. gARID1 contains several key residues for DNA binding, and its binding sequences are similar to those of the known ARID family proteins. The gARID1 binding sequences were positive cis-acting elements of the cwp1 promoter during both vegetative growth and encystation. We also found that gARID1 transactivated the cwp1 promoter through its binding sequences in vivo. Our results suggest that the ARID family has been conserved during evolution and that gARID1 is an important transactivator in regulation of the Giardia cwp1 gene, which is key to Giardia differentiation into cysts.

Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright

The B cell-restricted transcription factor, B cell regulator of Ig(H) transcription (Bright), up-regulates Ig H chain transcription 3- to 7-fold in activated B cells in vitro. Bright function is dependent upon both active Bruton's tyrosine kinase and its substrate, the transcription factor, TFII-I. In mouse and human B lymphocytes, Bright transcription is down-regulated in mature B cells, and its expression is tightly regulated during B cell differentiation. To determine how Bright expression affects B cell development, transgenic mice were generated that express Bright constitutively in all B lineage cells. These mice exhibited increases in total B220(+) B lymphocyte lineage cells in the bone marrow, but the relative percentages of the individual subpopulations were not altered. Splenic immature transitional B cells were significantly expanded both in total cell numbers and as increased percentages of cells relative to other B cell subpopulations. Serum Ig levels, particularly IgG isotypes, were increased slightly in the Bright-transgenic mice compared with littermate controls. However, immunization studies suggest that responses to all foreign Ags were not increased globally. Moreover, 4-wk-old Bright-transgenic mice produced anti-nuclear Abs. Older animals developed Ab deposits in the kidney glomeruli, but did not succumb to further autoimmune sequelae. These data indicate that enhanced Bright expression results in failure to maintain B cell tolerance and suggest a previously unappreciated role for Bright regulation in immature B cells. Bright is the first B cell-restricted transcription factor demonstrated to induce autoimmunity. Therefore, the Bright transgenics provide a novel model system for future analyses of B cell autoreactivity.

Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression

AbstractImmunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of “crippling” mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells. (Blood. 2004;104:3326-3334)