Different Isoforms of BSAP Protein Regulate the Expression of Activation-Induced Cytidine Deaminase (AID) in Normal and CLL B-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1901-1901
Author(s):  
Pablo J. Oppezzo ◽  
Gerard M. Dumas ◽  
Ana Ines Lalanne ◽  
Otto F. Pritsch ◽  
Francoise M. Vuillier ◽  
...  
Keyword(s):  
B Cells ◽  
Molecular Mechanisms ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Gene Promoter ◽  
Cd40 Ligand ◽  
Regulation Mechanism ◽  
Class Switch ◽  
Healthy Donors ◽  
Typical Event

Abstract Background: In B-cells somatic hypermutation (SHM) and class switch recombination (CSR) depend of AID expression, a typical event in germinal centers upon CD40 ligand (CD40L) stimulation. We recently showed, that in contrast to normal B-cells, AID is constitutively expressed in a majority of unmutated CLL patients (Oppezzo et al, Blood, 2003). Pax-5 gene, encodes for the B-specific activator protein (BSAP) an essential protein in B-cell development. BSAP has been shown to up-regulate AID gene expression, through binding to its promoter region, whereas inhibitor of differentiation Id-2 down-regulates it. To study the molecular mechanisms underlying AID regulation, we analyzed expression of BSAP and Id-2 transcripts in normal and CLL B-cells and correlated these results to expression of AID and CSR process. Results: B-cells from 6 healthy donors and 40 CLL patients were analyzed for Pax-5, Id-2, AID and CSR. Results show that CLL and normal B-cells not expressing AID exhibit diminished expression of BSAP and express a spliced variant of the Pax-5 gene (Pax-5 ΔEx8) displaying a deletion in the C-terminal-active domain. Upon CD40-L and IL-4 stimulation expression of AID transcripts and disappearance of the Pax-5 ΔEx8 transcripts was observed. Translation of complete Pax-5 and Pax-5/ΔEx8 isoforms and their binding to AID gene promoter have been demonstrated by Western Blot and EMSA assays, respectively. In contrast, CLL B-cells with constitutive AID expression and active CSR, constantly show increased levels of BSAP and absence of Pax-5ΔEx8 isoform. High expression of AID and BSAP is also correlated with a decrease of Id-2 transcripts. Conclusion: These results suggest that the presence of BSAP isoform deleted in exon 8 may interfere with the binding of the complete BSAP protein to AID promoter and down-regulate the expression of Pax-5a. Thus, we show for the first time a subtle regulation mechanism controlling the expression of AID protein through the differential splicing of a transcription factor as BSAP.

scholarly journals miR-181b negatively regulates activation-induced cytidine deaminase in B cells

2008 ◽  
Vol 205 (10) ◽  
pp. 2199-2206 ◽  
Author(s):  
Virginia G. de Yébenes ◽  
Laura Belver ◽  
David G. Pisano ◽  
Susana González ◽  
Aranzazu Villasante ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Bystander Effect ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Fine Tuning ◽  
Functional Screening ◽  
Activation Induced Cytidine Deaminase ◽  
Activated B Cells

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3′ untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

Mutator Genes UDG and AID Appear To Be Normal in Class-Switch Deficient and Clonally Homogeneous Waldenstrom’s Macroglobulinemias Having Either Germline or Hypermutated Clonotypic IgH VDJ.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1359-1359
Author(s):  
Jitra Kriangkum ◽  
Brian J. Taylor ◽  
Erin R. Strachan ◽  
Steven P. Treon ◽  
Michael J. Mant ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Splice Variants ◽  
Cytidine Deaminase ◽  
Essential Elements ◽  
Class Switching ◽  
Conserved Sequence ◽  
Class Switch ◽  
Mutator Genes

Abstract Clonotypic B cells of Waldenstrom’s macroglobulinemia (WM) are characterized as CD20+IgM+IgD+ cells that are usually somatically mutated in IgH VDJ but for some patients, the clonotypic IgH VDJ is germline (unmutated).For both mutated and unmutated clones, WM lack ongoing somatic hypermutation (SHM) and class switch recombination (CSR). This may be due to abnormalities in switching and/or mutator genes. To understand the nature of unswitched tumor B cells, uracil DNA glycosylase (UDG) and activation-induced cytidine deaminase (AID), the two essential elements for CSR, were analysed in WM. Analysis of 12 WM clones characterized by somatic hypermutation showed that the mutation profile of VH genes had normal transition/transversion ratios at C or G, and thus did not suggest UDG abnormalities. Expression of AID was determined by single stage RT-PCR. Out of 14 patients studied (2 unmutated and 12 mutated VH clones), two of them (WM1-01 and WM1-08,with mutation rates of 0% and 6.2% respectively) gave positive bands. In WM1-01, despite having a germline IgH VDJ, AID is consistently expressed in two bone marrow samples collected three years apart and from which the identical unmutated clonotypic VDJ sequence was isolated. Full-length (FL) AID transcripts of WM have a conserved sequence, thus ruling out the possibility of functional defects due to point mutation. In addition, detection of AID in an unmutated VH clone suggested that lack of SHM does not result from an inability to produce AID. In addition to FL transcripts, three other splice variants were identified in both patients. Single cell analysis indicated that only a small compartment (10% or less), not all, of clonotypic B cells expressed AID, and multiple isoforms may be detectable in individual cells. Whether these splice variants that contain truncated C-terminal ends play a role in the regulation of CSR in WM remains to be investigated. Splice variants, nevertheless, may not characterize tumor B cells since up to 10% of AID-expressing normal activated B cells (n=3) also carried them. In vitro activation of clonotypic WM B cells by CD40L and IL4, using conditions that induced CSR in normal B cells, did not yield detectable class switching in WM B cells. In cultures of B cells from WM, the number of non-clonal B cells increased but the clonotypic B cells did not appear to expand, as indicated by the reduction of clonotypic IgM transcript at 5-days of culture. Thus, as well as failing to undergo somatic mutation or class switching, WM tumor B cells appear unresponsive to CD40L+IL4. They may be fundamentally unresponsive to signals for class switching and their clonal expansion may depend upon alternate signaling pathways.

scholarly journals AID from bony fish catalyzes class switch recombination

2005 ◽  
Vol 202 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Vasco M. Barreto ◽  
Qiang Pan-Hammarstrom ◽  
Yaofeng Zhao ◽  
Lennart Hammarstrom ◽  
Ziva Misulovin ◽  
...  
Keyword(s):  
B Cells ◽  
Catalytic Domain ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Adaptive Immune System ◽  
Bony Fish ◽  
Class Switching ◽  
Class Switch ◽  
Switch Regions

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

scholarly journals LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1448-1455 ◽  
Author(s):  
Julia Rastelli ◽  
Cornelia Hömig-Hölzel ◽  
Jane Seagal ◽  
Werner Müller ◽  
Andrea C. Hermann ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Cd40 Ligand ◽  
Barr Virus ◽  
Class Switch ◽  
Epstein Barr

AbstractThe Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell–specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells.

scholarly journals A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification

2007 ◽  
Vol 204 (8) ◽  
pp. 1989-1998 ◽  
Author(s):  
Petra Langerak ◽  
Anders O.H. Nygren ◽  
Peter H.L. Krijger ◽  
Paul C.M. van den Berk ◽  
Heinz Jacobs
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Nuclear Antigen ◽  
Translesion Dna Synthesis ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Mismatch Recognition ◽  
Proliferating Cell

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine164 of proliferating cell nuclear antigen (PCNAK164) stimulates TLS. To determine the role of PCNAK164 modifications in somatic hypermutation, PCNAK164R knock-in mice were generated. PCNAK164R/K164R mutants are born at a sub-Mendelian frequency. Although PCNAK164R/K164R B cells proliferate and class switch normally, the mutation spectrum of hypermutated immunoglobulin (Ig) genes alters dramatically. A strong reduction of mutations at template A/T is associated with a compensatory increase at G/C, which is a phenotype similar to polymerase η (Polη) and mismatch repair–deficient B cells. Mismatch recognition, monoubiquitinated PCNA, and Polη likely cooperate in establishing mutations at template A/T during replication of Ig genes.

Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4029-4032 ◽  
Author(s):  
Pablo Oppezzo ◽  
Françoise Vuillier ◽  
Yuri Vasconcelos ◽  
Gérard Dumas ◽  
Christian Magnac ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Gene Product ◽  
Mode Of Action ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Lymphocytic Leukemia ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase

Abstract In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch μ DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.

Cessation of Somatic Hypermutation of Immunoglobulin Genes in Follicular Lymphomas after Isotype Switching Despite Continuous Expression of Activation-Induced Cytidine Deaminase (AID).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2412-2412
Author(s):  
Cristina Bertinetti ◽  
Julia Weiss ◽  
Hendrik Veelken
Keyword(s):  
B Cells ◽  
Success Rate ◽  
Mutation Frequency ◽  
Molecular Mechanisms ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Maturation Stage ◽  
Amino Acid Sequence Level ◽  
Activation Induced Cytidine Deaminase ◽  
Follicular Lymphomas

Abstract Follicular Lymphomas (FL) are considered the neoplastic counterpart of germinal center (GC) B cells due to their characteristic growth pattern, composition of a mixture of centroblasts and centrocytes, a GC gene expression profile, and ongoing acquisition of somatic mutations of their immunoglobulin (Ig) genes. A minority of FL has undergone Ig class switching from IgM to IgG or IgA at diagnosis. Based on PCR-mediated cloning of Ig heavy chain (IgH) genes with a success rate of 73%, Aarts et al. (Blood, 2000) have reported from an analysis of 30 FL cases that isotype-switched FL harbor significantly more IgH mutations than IgM-expressing cases, suggesting that the somatic hypermutation (SHM) machinery remains fully active even after class switch recombination (CSR) in FL. We have readdressed this question in 38 FL cases and compared their SHM pattern to 31 lymphomas of other types. 16 of these controls expressed IgM (including 5 MCL, 4 DLCL, and 3 CLL) and 15 IgG/A (including 13 myelomas). The Ig isotype and light chain (IgL) restriction of the lymphoma cells were identified by flow cytometry, and both clonal IgH and IgL transcripts were cloned by anchored PCR. This approach has a success rate of 98% (Bertinetti et al., Eur. J. Haematol., 2006). At the DNA (Table 1 and 2) and amino acid sequence level (not shown), the SHM frequency did not differ significantly between the 23 IgM-expressing and 15 class-switched FL. IgM-expressing FL, however, had more SHM than unswitched non-FL cases. In contrast, class-switched FL did not harbor more mutations than switched non-FL tumors. Although the molecular mechanisms are not yet fully understood, activation-induced cytidine deaminase (AID) has been identifed as an essential enzyme required for both SHM and CSR and is frequently expressed by GC lymphomas. To investigate whether downregulation of AID might be responsible for cessation of SHM after CSR in FL, we measured the AID expression in the FL biopsies by a semiquantitative RT-PCR. In contrast to our hypothesis, AID transcripts were present in both IgM- and IgG/A-expressing FL, and the median AID expression was even significantly higher in switched FL (p=0.014). These data indicate that despite their arrest in the GC maturation stage, FL behave similar to normal B cells by downregulating the SHM machinery after CSR. In addition, the data are consistent with a model in which intermediate expression of AID is sufficient for SHM, but higher levels are required to initiate CSR in FL. Subsequent cessation of SHM might be attributable to subcellular relocalisation of AID or decreased activity of AID cofactors, such as single-stranded binding protein (RPA) or protein kinase A (PKA). Median (range) of VH Mutations IgM IgG/A IgM versus IgG/A Comparison of mutation frequency of the clonal VH gene between non-switched and switched FL cases and between FL and non-FL cases FL (n=38) 32 (8–106) 38 (12–58) p=0.4 Non-FL (n=31) 18 (6–57) 25 (13–65) p=0.07 FL versus non-FL p=0.006 p=0.11 Median (range) of VL Mutations IgM IgG/A IgM versus IgG/A Comparison of mutation frequency of the clonal VL gene between non-switched and switched FL cases and between FL and non-FL cases FL (n=38) 14 (0–38) 18 (5–31) p=0.36 Non-FL (n=31) 5.5 (0–25) 14 (5–53) p=0.001 FL versus non-FL p=0.002 p=0.32

Hyper-IgM syndromes: a model for studying the regulation of class switch recombination and somatic hypermutation generation

2002 ◽  
Vol 30 (4) ◽  
pp. 815-818 ◽  
Author(s):  
A. Durandy
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Humoral Response ◽  
Cd40 Ligand ◽  
Class Switch ◽  
Hyper Igm Syndrome ◽  
Hyper Igm ◽  
Nuclear Factor Kb

Several genetic defects in class switch recombination, which lead to a hyper-IgM syndrome, have been described recently in humans. In addition to the well known role of CD40-ligand-CD40 interaction, these pathologies demonstrate definitively the requirement of CD40-mediated nuclear factor kB activation and the essential role of a recently described molecule, the activationinduced cytidine deaminase in an efficient humoral response, which includes class switch recombination and the production of high-affinity antibodies.

scholarly journals A primary immunodeficiency characterized by defective immunoglobulin class switch recombination and impaired DNA repair

2007 ◽  
Vol 204 (5) ◽  
pp. 1207-1216 ◽  
Author(s):  
Sophie Péron ◽  
Qiang Pan-Hammarström ◽  
Kohsuke Imai ◽  
Likun Du ◽  
Nadine Taubenheim ◽  
...  
Keyword(s):  
Dna Repair ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Cd40 Ligand ◽  
Dna Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Double Stranded Dna ◽  
Cd40 Activation

Immunoglobulin class switch recombination (CSR) deficiencies are rare primary immunodeficiencies, characterized by a lack of switched isotype (IgG, IgA, or IgE) production, variably associated with abnormal somatic hypermutation (SHM). Deficiencies in CD40 ligand, CD40, activation-induced cytidine deaminase, and uracil-N-glycosylase may account for this syndrome. We previously described another Ig CSR deficiency condition, characterized by a defect in CSR downstream of the generation of double-stranded DNA breaks in switch (S) μ regions. Further analysis performed with the cells of five affected patients showed that the Ig CSR deficiency was associated with an abnormal formation of the S junctions characterized by microhomology and with increased cell radiosensitivity. In addition, SHM was skewed toward transitions at G/C residues. Overall, these findings suggest that a unique Ig CSR deficiency phenotype could be related to an as-yet-uncharacterized defect in a DNA repair pathway involved in both CSR and SHM events.

Molecular analysis of a large cohort of patients with the hyper immunoglobulin M (IgM) syndrome

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 1881-1890 ◽  
Author(s):  
Wen-I Lee ◽  
Troy R. Torgerson ◽  
Michael J. Schumacher ◽  
Leman Yel ◽  
Qili Zhu ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Cd40 Ligand ◽  
Immunoglobulin M ◽  
Uracil Dna Glycosylase ◽  
Molecular Defects ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Cd40 Activation ◽  
Common Variable

AbstractThe hyper immunoglobulin M (IgM) syndrome (HIGM), characterized by recurrent infections, low serum IgG and IgA, normal or elevated IgM, and defective class switch recombination and somatic hypermutation, is a heterogenous disorder with at least 5 distinct molecular defects, including mutations of the genes coding for the CD40 ligand (CD40L) and IKK-gamma (NEMO) genes, both X-linked; and mutations of CD40, activation-induced cytidine deaminase (AICDA), and uracil-DNA glycosylase (UNG), associated with autosomal recessive HIGM syndromes. To investigate the molecular basis of HIGM, we determined the prevalence of mutations affecting these 5 genes in a cohort of 140 patients (130 males and 10 females). Those patients without a molecular diagnosis were subsequently evaluated for mutations of the following genes: inducible CO-stimulator molecule (ICOS), ICOS ligand (ICOSL), and if male, Bruton tyrosine kinase (Btk) and SLAM-associated protein (SAP/SH2D1A). We found mutations of CD40L in 98 males; AICDA in 4 patients (3 males, 1 female); UNG in one adult male; and Btk in 3 boys. Of the remaining 25 males, one infant with hypohidrotic ectodermal dysplasia had a mutation of NEMO. None of the remaining 33 patients (24 males/9 females) had mutations affecting CD40, ICOS, ICOSL, or SH2D1, and are best classified as common variable immune deficiency (CVID), although other genes, including some not yet identified, may be responsible.

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