Eric Pinaud

Transcription-targeted DNA deamination by the AID antibody diversification enzyme

Activation-induced cytidine deaminase (AID), which is specific to B lymphocytes, is required for class switch recombination (CSR)—a process mediating isotype switching of immunoglobulin—and somatic hypermutation—the introduction of many point mutations into the immunoglobulin variable region genes. It has been suggested that AID may function as an RNA-editing enzyme or as a cytidine deaminase on DNA. However, the precise enzymatic activity of AID has not been assessed in previous studies. Similarly, although transcription of the target immunoglobulin locus sequences is required for both CSR and somatic hypermutation, the precise role of transcription has remained speculative. Here we use two different assays to demonstrate that AID can deaminate specifically cytidines on single-stranded (ss)DNA but not double-stranded (ds)DNA substrates in vitro. However, dsDNA can be deaminated by AID in vitro when the reaction is coupled to transcription. Moreover, a synthetic dsDNA sequence, which targets CSR in vivo in a manner dependent on transcriptional orientation, was deaminated by AID in vitro with the same transcriptional-orientation-dependence as observed for endogenous CSR. We conclude that transcription targets the DNA deamination activity of AID to dsDNA by generating secondary structures that provide ssDNA substrates.

Long-range oncogenic activation of Igh-c-myc translocations by the Igh 3' regulatory region

B-cell malignancies, such as human Burkitt’s lymphoma, often contain translocations that link c-myc or other proto-oncogenes to the immunoglobulin heavy chain locus (IgH, encoded by Igh). The nature of elements that activate oncogenes within such translocations has been a long-standing question. Translocations within Igh involve DNA double-strand breaks initiated either by the RAG1/2 endonuclease during variable, diversity and joining gene segment (V(D)J) recombination, or by activation-induced cytidine deaminase (AID, also known as AICDA) during class switch recombination (CSR). V(D)J recombination in progenitor B (pro-B) cells assembles Igh variable region exons upstream of μ constant region (Cμ) exons, which are the first of several sets of CH exons (‘CH genes’) within a CH locus that span several hundred kilobases (kb). In mature B cells, CSR deletes Cμ and replaces it with a downstream CH gene. An intronic enhancer (iEμ) between the variable region exons and Cμ promotes V(D)J recombination in developing B cells. Furthermore, the Igh 3′ regulatory region (Igh3′RR) lies downstream of the CH locus and modulates CSR by long-range transcriptional enhancement of CH genes. Transgenic mice bearing iEμ or Igh3′RR sequences fused to c-myc are predisposed to B lymphomas, demonstrating that such elements can confer oncogenic c-myc expression. However, in many B-cell lymphomas, Igh–c-myc translocations delete iEμ and place c-myc up to 200 kb upstream of the Igh3′RR. Here we address the oncogenic role of the Igh3′RR by inactivating it in two distinct mouse models for B-cell lymphoma with Igh–c-myc translocations. We show that the Igh3′RR is dispensable for pro-B-cell lymphomas with V(D)J recombination-initiated translocations, but is required for peripheral B-cell lymphomas with CSR-associated translocations. As the Igh3′RR is not required for CSR-associated Igh breaks or Igh–c-myc translocations in peripheral B-cell lymphoma progenitors, we conclude that this regulatory region confers oncogenic activity by long-range and developmental stage-specific activation of translocated c-myc genes.

SYNERGIES BETWEEN REGULATORY ELEMENTS OF THE IMMUNOGLOBULIN HEAVY CHAIN LOCUS AND ITS PALINDROMIC 3' LOCUS CONTROL REGION

Transcriptional enhancers of the IgH locus include Eμ and four 3 ′ elements (Cα3 ′ , hs1‐2, hs3 and hs4 ), some of which are by themselves weak. We show that these weak elements behave as strong “co‐enhancers” when combined, and display a stage‐dependent activity which differs from that obtained when they are alone. Combinations mimicking the palindromic structure of the 3 ′ IgH region are particularly efficient. Noticeably in pre‐B cells, hs4 is boosted by the addition of elements previously considered inactive at this stage, hs1‐2 and hs3. Combinations of 3 ′ elements also strongly boost Eμ at all maturation stages, but inhibitory interactions occasionally occur between Eμ and incomplete 3 ′ combinations, indicating that full transcriptional activity is mainly achieved when all 5 ′ and 3 ′ partners play their respective roles.

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.

The 3′ IgH Locus Control Region Is Sufficient to Deregulate a c-myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to JH or within switch regions and always link c-myc to the 3′ IgH locus control region (3′ LCR). To test the hypothesis that the 3′ LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3′ LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220+IgM+IgD+ with the BL “starry sky” appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3′ IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.

Polymorphism of the human α1 immunoglobulin gene 3′ enhancer hs1,2 and its relation to gene expression

We studied the hs1,2 transcriptional enhancer identified downstream of the human α1 gene of the immunoglobulin H (IgH) locus, for which two different allelic configurations (a and b) were previously reported by Southern blotting. By using a polymerase chain reaction (PCR) method we amplified minisatellites within the hs1,2 core enhancer, with variable numbers of tandem repeats (VNTR) defining three ‘PCR alleles’α1A, α1B and α1C (including one, two and three repeats, respectively). Five different α1 h1,2 genotypes were encountered in a population of 513 donors, representing 13·8, 34·5, 49·7, 1·3 and 0·6% for the AA, BB, AB, AC and BC genotypes, respectively. Luciferase assays showed that increasing the number of minisatellites increased the transcriptional strength of the α1 hs1,2 enhancer. Simultaneous determination of Southern blot alleles and VNTR alleles only showed a partial linkage between both types of polymorphism, altogether defining at least six different allelic forms of the 3′α1 region. In conclusion, the present study further demonstrates the genetic instability of the 3′α region, for which multiple alleles have been generated through inversions and internal deletions and/or duplications. This study also strengthens the hypothesis that the polymorphism at the IgH 3′ regulatory region of the α1 gene could play a role in the outcome of diseases involving immunoglobulin secretion.

In Vivo Redundant Function of the 3′ IgH Regulatory Element HS3b in the Mouse

In the mouse, the regulatory region located at the 3′ end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igμ-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

Enhancers located in heavy chain regulatory region (hs3a, hs1,2, hs3b, and hs4) are dispensable for diversity of VDJ recombination.

Background: We examined the effect of deletion of the heavy chain regulatory region (RR) on VDJ recombination in B-cells. Results: V, D, and J usage is unaffected by the absence of the IgH RR. Conclusion: The IgH RR is dispensable for V(D)J diversity. Significance: This region only orchestrates IgH locus activity during late stages of B-cell differentiation. V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

The IgH 3′ regulatory region and its implication in lymphomagenesis

The 3′ regulatory region (3′RR) located downstream of the IgH gene is the master element that controls class switch recombination and sustains high‐level transcription at the plasma‐cell stage. This latter role suggests that the 3′RR may be involved in oncogene deregulation during the frequent IgH translocation events associated with B‐cell malignancies. A convincing demonstration of the essential contribution of 3′RR in lymphomagenesis has been provided by transgenic animal models. The mouse 3′RR shares a strong structural homology with the regulatory regions located downstream of each human Cα gene. Mouse models exploring the role of the 3′RR in B‐cell physiology and in malignancies should provide useful indications about the pathophysiology of human cell lymphocyte proliferation.

Interallelic class switch recombination can reverse allelic exclusion and allow trans-complementation of an IgH locus switching defect

The predominant path of immunoglobulin class switch recombination follows the paradigm of intra‐chromosomal deletion enabling expression of another heavy chain instead of µ and δ. This was, however, challenged by observations of inter‐allelic class switch recombination in rabbit or mouse IgG3‐ or IgA‐producing B cells. Assuming that the conditions of inter‐chromosomal exchange are likely present at any target S regions in stimulated B cells, we explored trans‐association of VH and C genes in a model allowing all C genes to be checked simultaneously. Heterozygous mutant mice are thus studied, which carry one non‐functional IgH allele inactivated by a non‐translatable mutation of VDJ‐CH transcripts, while the functional allele is deficient for class switching due to a truncated 3′regulatory region. A fair level of switching to all Ig classes is restored in heterozygous mice despite the fact that cis‐recombination is either non productive on one allele or deficient on the other. Molecular evidence at the DNA level of trans‐CSR to IgG3 was demonstrated by cloning and sequencing Sµ‐Sγ3 hybrid junctions. These data demonstrate that inter‐allelic recombination may broadly rescue the production of various class‐switched isotypes and allow complementation between mutations located at both ends of the IgH constant gene cluster.