Alexis Saintamand

The IgH 3′ regulatory region controls somatic hypermutation in germinal center B cells

Somatic hypermutation in variable heavy chain rearranged regions is abrogated in the absence of the 3′ regulatory region enhancer, whereas transcription rate in the Ig heavy chain is only partially reduced.

Elucidation of the enigmatic IgD class-switch recombination via germline deletion of the IgH 3′ regulatory region

Unlike classical class-switch recombination, class switching to IgD occurs independently of the IgH 3′ regulatory region.

Sequential activation and distinct functions for distal and proximal modules within the IgH 3' regulatory region.

Significance The immunoglobulin heavy chain (IgH) 3′regulatory region (3′RR) fine-tunes IgH gene expression during B cell development. One singularity of this region is its quasi-palindromic structure conserved in the 3′RR of other species. By comparing previous mouse knockout (KO) models (3′RR- and hs3b-4 KO) to a novel mutant devoid of the quasi-palindrome (3′PAL KO), we highlighted common features and differences that specify two distinct regulatory entities: (i) the distal module (hs4) is sufficient for normal IgH expression up to the naïve B cell stage; (ii) during B-cell activation, the proximal module (quasi-palindrome) is important for both class switch recombination and somatic hypermutation; and (iii) in plasma cells, the quasi-palindrome is required for robust transcription of the IgH locus. As a master regulator of functional Ig heavy chain (IgH) expression, the IgH 3′ regulatory region (3′RR) controls multiple transcription events at various stages of B-cell ontogeny, from newly formed B cells until the ultimate plasma cell stage. The IgH 3′RR plays a pivotal role in early B-cell receptor expression, germ-line transcription preceding class switch recombination, interactions between targeted switch (S) regions, variable region transcription before somatic hypermutation, and antibody heavy chain production, but the functional ranking of its different elements is still inaccurate, especially that of its evolutionarily conserved quasi-palindromic structure. By comparing relevant previous knockout (KO) mouse models (3′RR KO and hs3b-4 KO) to a novel mutant devoid of the 3′RR quasi-palindromic region (3′PAL KO), we pinpointed common features and differences that specify two distinct regulatory entities acting sequentially during B-cell ontogeny. Independently of exogenous antigens, the 3′RR distal part, including hs4, fine-tuned B-cell receptor expression in newly formed and naïve B-cell subsets. At mature stages, the 3′RR portion including the quasi-palindrome dictated antigen-dependent locus remodeling (global somatic hypermutation and class switch recombination to major isotypes) in activated B cells and antibody production in plasma cells.

IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3′ regulatory region super-enhancer

IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3′ regulatory region super-enhancer

The IgH 3’ regulatory region and c-myc-induced B-cell lymphomagenesis

Deregulation and mutations of c-myc have been reported in multiple mature B-cell malignancies such as Burkitt lymphoma, myeloma and plasma cell lymphoma. After translocation into the immunoglobulin heavy chain (IgH) locus, c-myc is constitutively expressed under the control of active IgH cis-regulatory enhancers. Those located in the IgH 3 regulatory region (3RR) are master control elements of transcription. Over the past decade numerous convincing demonstrations of 3RRs contribution to mature c-myc-induced lymphomagenesis have been made using transgenic models with various types of IgH-c-myc translocations and transgenes. This review highlights how IgH 3RR physiological functions play a critical role in c-myc deregulation during lymphomagenesis.

Comment on “IgH Chain Class Switch Recombination: Mechanism and Regulation”

Class switch recombination (CSR) is a deletional DNA recombination occurring in IgM+IgD+ B cells and overwhelmingly recognized for thereby generating IgG, IgA, and IgE Abs with the same antigenic specificity but new effector functions. In an interesting and well documented paper, Stavnezer and

3′RR targeting in lymphomagenesis: a promising strategy?

During precursor B-cell differentiation, heavy (H) and light chain genes of an immunoglobin (Ig) molecule are somatically assembled from germline DNA. This V(D)J recombination process occurs in the bone marrow prior to antigenic challenge (Fig. 1). In germinal centers, variable (V) regions become the target of somatic hypermutation (SHM) in activated B-cells generating high-affinity Ig (Fig. 1). In mature B-cells, class-switch recombination (CSR) deletes the constant (C) μ region and replaces it with a downstream CH gene. This enables B-cells to express various Ig isotypes without affecting antigen specificity (Fig. 1). Once activated, B-cells differentiate into Ig-secreting plasma cells. During B-cell development, V(D)J recombination, SHM, CSR and Ig synthesis are coupled with transcriptional accessibility of the IgH loci. IgH transcription is controlled by complex functional interactions of multiple enhancers, promoters and insulators spread among the 2.5 megabases of the locus. Among them, the 3′ regulatory region (3′RR) stands out as a major player during late stages of B-cell maturation (i.e., SHM, CSR and Ig synthesis).1,2 Figure 1. DNA recombination and mutations occur during B-cell maturation. RAG-induced (during VDJ recombination) and AID-induced (during CSR and SHM) DNA breaks are potential sites of oncogene translocations. The IgH 3′RR may act as an oncogene deregulator ... Ongoing recombination and mutation all along B-cell development make the IgH locus a hotspot for translocations. Numerous lymphomas are marked by proto-oncogene translocation into the IgH locus. Cyclin D1 and Bcl-2 translocations, found respectively in mantle cell lymphoma and follicular lymphoma, take place during the V(D)J recombination. Cyclin D1/D3 or c-maf translocations observed in myeloma are obviously linked to CSR. c-myc translocation, the typical hallmark of Burkitt lymphoma, is linked to SHM or CSR. The mouse 3′RR, located downstream of the IgH Cα gene, shares a strong structural homology with the regulatory regions located downstream of each human IgH Cα gene (Cα1 and Cα2). Mouse models exploring the role of the 3′RR in B-cell physiology and in B-cell malignancies should provide useful indications about the pathophysiology of human B-cell proliferations. Convincing demonstration of the key contribution of the 3′RR in mature B-cell lymphomagenesis has been done by transgenic animal models. c-myc-3′RR transgenics developed Burkitt lymphoma-like proliferation,3 and the knock-in of a 3′RR cassette upstream of the endogenous c-myc gene induced B-cell lymphomas.4 Interestingly the phenotype of lymphoproliferations induced by the c-myc-3′RR transgene is affected by the presence of associated mutations in key cell cycle dependent genes such as p53 or Cdk4.5 Knock-out models have clarified the functions of the 3′RR as essential for high-rate IgH transcription at the plasma cell stage.2 3′RR may thus be a potent activator of IgH-translocated oncogene transcription, even when the breakpoints lie several hundred kb away from the 3′RR. Long-range interactions between the 2 regions of chromatin, through formation of a loop structure, constitute an important mechanism of normal and abnormal gene transcription regulation by the 3′RR. Studies have reported interactions between the 3′RR and the IgH variable region in normal and lymphomagenetic contexts. Therefore, targeted inhibition of the 3′RR could theoretically provides a therapeutic strategy for the treatment of a wide range of mature B-cell lymphomas. However, the first step before considering the 3′RR as a potential suitable target for anti-lymphoma pharmacological therapy is to demonstrate the innocuousness of such an approach, and notably the absence of potent adverse effect on normal immune and inflammatory B-cell mediated responses. Induction of negative alterations on the physiological anti-lymphoma immune or inflammatory networks would be obviously a counterproductive approach. We have recently tested this prerequisite by investigating the in vivo pristane-induced inflammatory response in 3′RR-deficient BALB/c and wt BALB/c mice.6 The lack of the 3′RR in BALB/c mice has no significant effect on the incidence, the kinetic of development and the cellular composition (IgM+IgD+ B-cells, CD4+ T cells, CD8+ T cells, monocyte/macrophage cells) of peritoneal ascites. Moreover ascite pro- and anti-inflammatory cytokine levels (IL-6, IL-21, IL-12/23, TNF-α IL-10, interferon-γ) are unaffected by the 3′RR-deficiency. Thus, a fully functional 3′RR is dispensable for the efficient recruitment of immune cells and the development of a normal inflammatory response in the in vivo pristane-induced inflammatory model. In conclusion, the 3′RR is considered as a major lymphoma oncogene deregulator,3-5 and its deletion has no effect on immune and inflammatory responses in the pristane mouse model. It is, thus, tempting to speculate that the 3′RR might be considered as a potential suitable target for anti-lymphoma pharmacological therapy without significant impact on the normal immune and inflammatory networks. Previous results have reported that the 3′RR is a sensitive immunological target and that 3′RR activation and transcriptional activity can be altered by a diverse range of chemicals, including ones with anti-carcinogenic properties such as isothiocyanates,7 strengthening the hypothesis that altering 3′RR activity by chemicals could modulate the occurrence and severity of lymphomas. A limitation of the pristane mouse model is that inflammation is restricted to the peritoneal cavity. It is of evidence that other mouse models of inflammatory reactions must be tested before definitive validation of this hypothesis. Finally given the strong sequence homology between human and mouse 3′RR enhancers, mouse models could reveal useful tools for an in vivo study of lymphoma treatments based on IgH 3′RR down-regulation.

Efficient role of IgH 3' regulatory region deficient B-cells in the development of oil granulomas

Functional B-cells are essential for the formation of oil granulomas. The IgH 3′ regulatory region (3′RR) activates important check-points during B-cell maturation. We investigated if 3′RR-deficient B-cells remain efficient to develop oil granulomas in response to pristine. B-cells expressing an IgH 3′RR-deficient allele were similarly recruited to wild type allele expressing B-cells in the granuloma. No differences were observed between 3′RR-deficient mice and control mice for granuloma numbers, cellular composition and ability to express mRNA transcripts for several pro- and anti-inflammatory cytokines. Altogether these results suggest a normal role for 3′RR-deficient B-cells in the development of an acute B-cell-mediated inflammatory response.