Reiko Shinkura

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-κb-inducing kinase

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyers patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT β-receptor (Ltβr, encoded by Ltbr ) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 (ref. 11) is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-κb-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-κb activation induced by an excess of Ltβr. Our observations agree with previous reports that Ltβr-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-κb activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6; ref. 13), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr ( ref. 12), Cd40 (Refs 14, 15), Cd30 (Refs 16, 17) and Ltβr (refs 18, 19, 20, 21). The phenotypes of aly/aly mice are more severe than those of Ltbr–/– mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms.

Separate domains of AID are required for somatic hypermutation and class-switch recombination

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM). Mutants with changes in the C-terminal region of AID retain SHM but lose CSR activity. Here we describe five mutants with alterations in the N-terminal region of AID that caused selective deficiency in SHM but retained CSR, suggesting that the CSR and SHM activities of AID may dissociate via interaction of CSR- or SHM-specific cofactors with different domains of AID. Unlike cells expressing C-terminal AID mutants, B cells expressing N-terminal AID mutants had mutations in the switch μ region, indicating that such mutations are generated by reactions involved in CSR but not SHM. Thus, we propose that separate domains of AID interact with specific cofactors to regulate these two distinct genetic events in a target-specific way.

The influence of transcriptional orientation on endogenous switch region function

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) takes place between large switch (S) regions that precede exons of the constant region. The precise functions of the S region are controversial, although transcription of the S region targets CSR. We have tested the effects of deletion, inversion and replacement of the endogenous 12-kilobase Sγ1 region on CSR in vivo. Here we show that Sγ1 is required for CSR, that CSR is effected by a 1-kilobase sequence that generates a G-rich transcript, and that inversion of Sγ1 or the G-rich sequence decreases CSR. We conclude that Sγ1 function is dependent on orientation and lacks an absolute requirement for common S region motifs. We propose that single-stranded DNA stabilized by transcription-dependent, higher order structures is a primary substrate of CSR.

Mice carrying a knock-in mutation of Aicda resulting in a defect in somatic hypermutation have impaired gut homeostasis and compromised mucosal defense

To elucidate the specific role of somatic hypermutation (SHM) in mucosal immunity, we generated mice carrying a knock-in point mutation in Aicda, which encodes activation-induced cytidine deaminase (AID), an enzyme essential to SHM and class-switch recombination (CSR). These mutant AIDG23S mice had much less SHM but had normal amounts of immunoglobulin in both serum and intestinal secretions. AIDG23S mice developed hyperplasia of germinal center B cells in gut-associated lymphoid tissues, accompanied by expansion of microflora in the small intestine. Moreover, AIDG23S mice had more translocation of Yersinia enterocolitica into mesenteric lymph nodes and were more susceptible than wild-type mice to oral challenge with cholera toxin. Together our results indicate that SHM is critical in maintaining intestinal homeostasis and efficient mucosal defense.

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac‐mediated actin‐based motility

The Wiskott–Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin‐cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene‐targeted mutation to examine its role in murine development and in actin assembly. WAVE2‐deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2‐deficient embryonic stem cells displayed normal proliferation, whereas WAVE2‐deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac‐mediated actin polymerization. These results imply a non‐redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin‐based processes downstream of Rac that are essential for cell movement.

Growth Retardation, Early Death, and DNA Repair Defects in Mice Deficient for the Nucleotide Excision Repair Enzyme XPF

ABSTRACT Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5′ to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.

Discovery of activation-induced cytidine deaminase, the engraver of antibody memory.

Discovery of activation-induced cytidine deaminase (AID) paved a new path to unite two genetic alterations induced by antigen stimulation; class switch recombination (CSR) and somatic hypermutation (SHM). AID is now established to cleave specific target DNA and to serve as engraver of these genetic alterations. AID of a 198-residue protein has four important domains: nuclear localization signal and SHM-specific region at the N-terminus; the alpha-helical segment (residue 47-54) responsible for dimerization; catalytic domain (residues 56-94) shared by all the other cytidine deaminase family members; and nuclear export signal overlapping with class switch-specific domain at the C-terminus. Two alternative models have been proposed for the mode of AID action; whether AID directly attacks DNA or indirectly through RNA editing. Lines of evidence supporting RNA editing hypothesis include homology in various aspects with APOBEC1, a bona fide RNA editing enzyme as well as requirement of de novo protein synthesis for DNA cleavage by AID in CSR and SHM. This chapter critically evaluates DNA deamination hypothesis and describes evidence to indicate UNG is involved not in DNA cleavage but in DNA repair of CSR. In addition, UNG appears to have a noncanonical function through interaction with an HIV Vpr-like protein at the WXXF motif. Taken together, RNA editing hypothesis is gaining the ground.

B cell-specific and stimulation-responsive enhancers derepress Aicda by overcoming the effects of silencers

Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch–inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5′ promoter region. The 5′ upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-κB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell–specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell–specific and stimulation-responsive elements and ubiquitous silencers.

The C-terminal region of activation-induced cytidine deaminase is responsible for a recombination function other than DNA cleavage in class switch recombination

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3–2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.