Dependence of antibody gene diversification on uracil excision

Noia, Javier M. Di; Williams, Gareth T.; Chan, Denice T. Y.; Buerstedde, Jean-Marie; Baldwin, Geoff; Neuberger, Michael S.

doi:10.1084/jem.20071768

Cited by 63 publications

(51 citation statements)

References 52 publications

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“…The IRCM Animal Ethics Committee approved all animal handling. Purification of resting B cells from splenic lymphocytes by CD43-depletion, retroviral infection and analysis of isotype class switching to IgG1 has been described 58 . AID-GFP variants and GFP-AID were subcloned as EcoRI-NotI fragments into the pMXs retroviral vector.…”

Section: Methodsmentioning

confidence: 99%

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Patenaude

Orthwein

et al. 2009

Nat Struct Mol Biol

133

175

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The enzyme Activation Induced Deaminase (AID) triggers antibody diversification in B-cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, its nuclear entry requiring active import mediated by a conformational nuclear localization sequence (NLS). We also identify a determinant for AID cytoplasmic retention in its Cterminus, which hampers diffusion to the nucleus, competes with nuclear import and is critical for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.3

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Section: Methodsmentioning

confidence: 99%

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Patenaude

Orthwein

et al. 2009

Nat Struct Mol Biol

133

175

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“…The mice were infected (24-h incubation) with hAID-encoding retroviruses that had been harvested from Plat-E cells 36 h after they had been transfected using Genejuice (Novagen) with pMx-GFP-derived plasmids in which hAID expression (along with that of GFP from an IRES, to provide an internal control) is directed from the retroviral LTR (27). CSR to IgG1 was analyzed by flow cytometry 3 days after retroviral infection, gating on live B cells that amounted to 5 to 7% of events, regardless of the identity of the retroviral construct (26). Procedures involving the use of animals were carried out under UK Home Office Project License 80/2226.…”

Section: Methodsmentioning

confidence: 99%

“…B cells were purified from AID Ϫ/Ϫ mice and cultured in the presence of LPSϩIL4, as previously described (26). The mice were infected (24-h incubation) with hAID-encoding retroviruses that had been harvested from Plat-E cells 36 h after they had been transfected using Genejuice (Novagen) with pMx-GFP-derived plasmids in which hAID expression (along with that of GFP from an IRES, to provide an internal control) is directed from the retroviral LTR (27).…”

Section: Methodsmentioning

confidence: 99%

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Geisberger

Rada²,

Neuberger³

2009

Proc. Natl. Acad. Sci. U.S.A.

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The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.activation-induced deaminase ͉ antibody diversification ͉ immunoglobulin class switching ͉ exportin ͉ nuclear transport A ctivation-induced deaminase (AID) plays a central role in antibody gene-diversification, triggering both somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating deoxycytidine residues within the Ig locus to yield deoxyuridine (1, 2).AID is largely found in the cytoplasm of activated B cells but shuttles between cytoplasm and nucleus (3-6). The C-terminal amino acids of AID comprise a nuclear-export sequence (NES): removal of this NES causes AID to be restricted to the nucleus, whereas its addition to a heterologous nuclear protein drives export of the fusion protein into the cytoplasm (3-5). The sequence of the AID C-terminus conforms well to the consensus established for substrates of the Crm1-dependent export machinery: in keeping with this, export mediated by the AID NES is sensitive to leptomycin B.Analysis of both clinical and contrived mutations in AID have revealed that the AID C-terminal region is also essential for its function in CSR, although not in SHM (7-9). The C-terminal mutations that interfere with AID's ability to potentiate CSR do not compromise its ability to deaminate deoxycytidine in biochemical or bacterial genetic assays. Thus, as previously suggested by others, either the nuclear/cytoplasmic shuttling of AID must be specifically required for CSR or, alternatively, factors specific for CSR must interact with the C-terminal region of AID in an area overlapping the NES (4, 5, 7-9).It was with a view to discriminating these possibilities that we embarked on a refined mutagenic dissection of the AID NES and investigated the ability of heterologous NESs to substitute for the AID NES. Our results suggest that although nuclear export is likely necessary for CSR, this is not the sole function of the AID C-terminal region, with the data raising the possibility that successful CSR depends on the precise quality of AID binding to exportin or a related protein. Results NES Function and CSR Correlate in Alanine-Scanning Mutagenesis.To ascertain whether there was a correlation between export activity and CSR, we performed an alanine-scanning mut...

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“…The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).…”

mentioning

confidence: 99%

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Shalhout

Haddad

Sosin

et al. 2014

Molecular and Cellular Biology

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d Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG ؊/؊ cells, uracil levels increase by 11-to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG ؊/؊ splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. When B lymphocytes are activated through antigen presentation, they acquire hypermutations in the immunoglobulin (Ig) genes, facilitating affinity maturation of antibodies. An enzyme, activation-induced deaminase (AID), initiates these events by converting cytosines in DNA to uracil (1-4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable domain (known as somatic hypermutations [SHMs]; reviewed in references 5 and 6). Generation of uracils is also the first step in the creation of strand breaks in the switch regions of Ig genes, leading to the replacement of the constant domain with other domains such as ␥, in a process called class-switch recombination (CSR; reviewed in reference 7). AID also binds near the transcription start sites of a large number of actively transcribed genes (8) and mutates a number of additional genes, including those encoding BCL-6, MYC, PAX-5, and PIM1 (9-12). The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).There is a strong connection between expression of AID and cancers in animals. Constitutive expression of AID in mice causes T cell cancers (18). Many human...

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Dependence of antibody gene diversification on uracil excision

Cited by 63 publications

References 52 publications

Active nuclear import and cytoplasmic retention of activation-induced deaminase

Active nuclear import and cytoplasmic retention of activation-induced deaminase

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

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