The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schoetz, U.
Right arrow Articles by Buerstedde, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schoetz, U.
Right arrow Articles by Buerstedde, J.-M.
The Journal of Immunology, 2006, 177: 395-400.
Copyright © 2006 by The American Association of Immunologists

E2A Expression Stimulates Ig Hypermutation1

Ulrike Schoetz, Manuela Cervelli2, Yan-Dong Wang, Petra Fiedler and Jean-Marie Buerstedde3

Institute of Molecular Radiobiology, GSF National Research Center for Environment and Health, Neuherberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ig hypermutation is limited to a region of ~2 kb downstream of the transcription start sites of the Ig loci. The process requires transcription and the presence of Ig enhancer sequences, and is initiated by the activation-induced cytidine deaminase (AID)-mediated deamination of cytidine bases. It remains unknown why AID causes mutations selectively in the Ig genes and not in most other transcribed loci of B cells. In this study, we report that the inactivation of the E2A gene strongly reduces the rate of Ig L chain mutations in the chicken B cell line DT40 without affecting the levels of surface Ig or AID expression. The defect is complemented by the expression of cDNAs corresponding to either of the two E2A splice variants E12 or E47. The results suggest that E2A-encoded proteins enhance Ig hypermutation by recruitment of AID to the Ig loci.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cells diversify the rearranged V(D)J segments within their L and H chain Ig loci by frequent nucleotide substitutions leading to the emergence of Abs with increased Ag affinity (1). Ig hypermutation distinguishes itself from other known hypermutation states by being limited to a region of ~2 kb downstream of the Ig gene transcription start sites (2). Although transcription of the Ig genes is required for hypermutation, the vast majority of all transcribed genes of B cells do not accumulate mutations at detectable frequency. Nevertheless, mutations, most likely reflecting an increased mutation rate and deregulated hypermutation activity, have been found in some non-Ig genes of B cells (3). Analysis of artificial transgenes in hypermutating B cells indicates that the Ig promoter can be replaced by other promoters without an effect on hypermutation, but that some of the Ig enhancer sequences are indispensable for the mutation activity (4, 5). Interestingly, the incidental introduction of an E-box transcription factor recognition sequence strongly increased the hypermutation activity of a transgene without affecting its transcription level (6).

Ig hypermutation as well as Ig gene conversion and switch recombination require expression of the activation-induced cytidine deaminase (AID)4 gene (7, 8, 9, 10). AID is believed to initiate hypermutation and recombination by the deamination of cytidine in the Ig loci (11). Surprisingly, AID expression in fibroblasts leads to a high mutation rate in non-Ig transgenes (12), suggesting that the restriction of hypermutation to the Ig loci is B cell specific or related to chromatin configuration. Nonphysiologic expression of AID in T cells increased the mutation rate in the same subset of non-Ig genes that were previously shown to be mutated in B cells (13).

The critical role of the cis-acting Ig enhancer sequence led to the proposal that one or more enhancer binding transcription factors recruit a mutator protein to the Ig loci after binding to the enhancer (5). One of the best candidates for such a factor is the E2A-encoded E47 protein, which can bind to artificially introduced E-box sequences (6) as well as to natural E-box motifs in the Ig enhancers. E2A-encoded proteins serve multiple functions in B cell commitment and differentiation most likely by regulating the transcription activity of genes (reviewed in Ref. 14).

To determine whether E2A influences Ig hypermutation, we decided to disrupt the gene in a hypermutating variant of the B cell line DT40 and compare the mutation frequencies in E2A-positive and -negative cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Construction of the E2A knockout constructs and cDNA expression vectors

The full-coding E12 and E47 cDNAs and the exon-intron structure of the E2A locus have been described previously (15). The target arms for the E2A knockout constructs were amplified by long-range PCR using genomic DNA from DT40 as template (Fig. 1A). The primer included restriction sites attached at the 5' end to facilitate cloning of the PCR fragments. Primers used for the amplification of the 5' arm and the 3' arm of the pE2Absr construct were E2A1/E2A2 and E2A3/E2A4, respectively (see Table Ifor all primer sequences). Primers used for the amplification of the 5' arm and the 3' arm of the pE2Agpt construct were E2A1/E2A5 and E2A6/E2A7, respectively. The arms were cloned upstream and downstream of the floxed drug resistance marker cassettes (16). The constructs were linearized by NotI before transfection. E12 and E47 full-length cDNAs were isolated from the riken1 bursal cDNA library (17) and their entire sequence was verified by primer walks. The E12 and E47 expression constructs were made by cloning the corresponding cDNAs downstream of the beta-actin promoter and upstream of an internal ribosomal entry site bsr sequence.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 1. E2A gene disruption. A, Aligned maps of the chicken E2A locus, the targeting constructs and the disrupted locus after targeting integration of the constructs and marker excision. B, Flow chart showing the generation of the E2A knockout and recomplemented clones

 

View this table:
[in this window]
[in a new window]
  		Table I. Sequences of primers used in PCR experiments

 
Generation of the E2A mutant clones

To generate the E2A mutant clones, we used the surface (s)Ig+ DT40 clone AIDR{psi}V– (18, 19), a mutant in which all pseudogenes of the rearranged L chain allele had been deleted. The pseudogene deletion stopped gene conversion and activated hypermutation at the L chain locus. This hypermutation activity is stably maintained even during prolonged culture of the clone as long as AID is expressed (18, 19). The use of the sIg+ clone AIDR{psi}V– is advantageous for studying Ig hypermutation, because the hypermutation rate can be estimated by measuring loss of sIg during the propagation of subclones.

The pE2Absr construct was transfected into the AIDR{psi}V– clone (18), and blasticidin-resistant colonies having integrated the construct by targeted integration were identified using a sense primer derived from the E2A locus upstream of the 5' target arm (E2A8) and an antisense primer derived from the respective drug resistance marker (Bs1 for bsr and Gp1 for gpt). Targeting efficiency after transfection of the AIDR{psi}V– clone by the pE2Absr construct was ~20%. One of the heterozygous clones was retained and named AIDR{psi}V–E2A+/–. As a homozygous E2A disrupted clone could not be identified after transfection of AIDR{psi}V–E2A+/– by a targeting construct that differed from pE2Absr only by including the gpt gene instead of the bsr gene, a new construct was made, which specifically targets the remaining wild-type allele of the heterozygous E2A mutant. Transfection of this pE2Agpt construct into AIDR{psi}V–E2A+/– yielded the homozygous E2A disrupted clone AIDR{psi}V–E2A–/–.

Cell culture

Cell culture, transfection, selection of stable transfectants, and marker recycle by transient Cre induction were performed as previously described (9).

Quantification of E2A and AID mRNA levels by semiquantitative PCR

AID mRNA was determined as previously described (9) using the primer pair AI1/AI2. The same protocol was used for the quantification of E12 and E47 mRNA levels. Expression levels for E12 and E47 were detected by the primer pairs E2A9/E2A10 and E2A9/E2A11, respectively. To determine the level of Ig L chain transcription, the L chain C region was amplified using the primer pair Cl1/Cl2. Amplification of transcripts of the housekeeping gene Elongation Factor 1{alpha} served as a control and was conducted using the primer pair EF6/EF7.

Ig mutation assay

Quantification of sIg expression by FACS was performed as described previously (9), except that goat anti-mouse IgG ((H+L)-RPE; Southern Biotechnology Associates) was used for second Ab staining.

FACS measurements were conducted with at least 24 subclones. The average percentages of sIg– cells for the mutant and control cell lines are calculated as mean values of all clonally derived subclones to account for fluctuation effects (20).

A low number of cells and a few subclones had apparently lost AID expression as indicated by the loss of the coupled GFP expression. This reflects most likely the excision of the floxed AID-internal ribosomal entry site-GFP transgene cassette by leaky Cre recombinase expression. Because a lack of AID expression stops Ig hypermutation (9), GFP-negative cells were excluded from the analysis. Subclones of AIDR{psi}V–E2ARtE12 and AIDR{psi}V–E2ARtE47 with >85% sIg– cells were excluded from the study, too, because these subclones were most likely derived from an already sIg– clone (Table II).


View this table:
[in this window]
[in a new window]
  		Table II. Percentages of events falling into the slgM– gates for individual subclones

 
Sequence analysis

Sequence analysis of the L chain VJ regions has been described previously (9, 18, 19). One subclone for each of the cell lines AIDR{psi}V–E2A–/–, AIDR{psi}V–E2ARtE12, and two for AIDR{psi}V–E2ARtE47 were maintained in culture for 6 wk. DNA was amplified by PCR with the primer pair VL1/2. The primer VL3 was used for sequencing. Cloning was performed in pUC119 vector after digestion with HindIII and XbaI.

The consensus sequence of all sequences from each subclone was taken as the likely sequence of the precursor cell of the subclone. Differences in the subclone sequences in comparison to the precursor cell sequence are regarded as mutations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Disruption of the E2A gene and mutant complementation by E12 and E47 cDNA expression

The 5' and 3' arms of the knockout constructs were designed to inactivate the E2A gene by the deletion of the exons encoding codons 142–463 in the case of pE2Absr and codons 172–353 in the case of pE2Agpt (Fig. 1A). The sIg+ DT40 variant AIDR{psi}V– (18) was chosen as the progenitor clone of the study, because it diversifies its rearranged Ig L chain locus by hypermutation, expresses AID under a constitutive promoter, and can be induced for Cre recombinase. Stepwise transfections of the E2A knockout constructs, identification of targeted transfectants, and excision of the drug resistance marker cassettes by Cre induction yielded the homozygous E2A mutant clone AIDR{psi}V–E2A–/– (Fig. 1B). To complement the E2A disruption, AIDR{psi}V–E2A–/– was transfected either by an E12 or an E47 cDNA expression vector, yielding the clones AIDR{psi}V–E2ARtE12 and AIDR{psi}V–E2ARtE47. The status of E2A expression in the wild-type, E2A mutant, and E2A-complemented clones was confirmed by RT-PCR (Fig. 2).


Figure 2
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 2. E12, E47, AID, and C{lambda} mRNA levels measured by semiquantitative RT-PCR. The housekeeping gene Elongation Factor 1{alpha} was amplified as an internal standard.

 
E2A does not influence Ig L chain transcription and AID expression

FACS analysis indicated that the majority of cells in all isolated clones remained sIg+ and that differences in E2A expression did not measurably influence the sIg levels of sIg+ cells (Fig. 3A, data not shown). Amplification of Ig L chain transcripts by semiquantitative RT-PCR using primers for the C region (Fig. 2) confirmed that the differences of E2A expression did not alter the steady-state level of the Ig L chain mRNA, suggesting that E2A proteins are not required for Ig L chain transcription in the analyzed DT40 clones. RT-PCR using AID cDNA specific primers demonstrated furthermore that the clones AIDR{psi}V–, AIDR{psi}V–E2A–/–, and AIDR{psi}V–E2ARtE12 expressed roughly equivalent amounts of AID mRNA, whereas the clone AIDR{psi}V–E2ARtE47 seems to express reduced levels of AID mRNA (Fig. 2). AID is expressed as an artificial cDNA under the control of a beta-actin promoter in the AIDR{psi}V– progenitor clone and all mutant clones derived from this clone, making it unlikely that its expression levels are influenced by E2A. In addition, AID expression levels can be estimated based on the expression of the coexpressed GFP, and no differences in the average GFP expression could be detected in E2A-positive and -negative clones (Fig. 3A).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. sIgM expression analysis of E2A–/– and control clones. A, FACS anti-IgM staining profiles of representative subclones derived from initially sIg+ clones. B, Average percentages of events falling into sIg– gates based on the measurement of at least 24 subclones of the cell lines AID–/–{psi}V–, AIDR{psi}V–, AIDR{psi}V–E2A+/–, AIDR{psi}V–E2A–/–, AIDR{psi}V–E2ARtE12, and AIDR{psi}V–E2ARtE47.

 
E2A-negative clones display strongly decreased rates of sIg loss

The fact that all clones included in the study remained predominantly sIg+ allowed estimation of the rate of deleterious Ig L chain mutations by measuring the appearance of sIg– populations (18). The appearance of sIg– populations was measured in parallel in at least 24 subclones of the following clones: AID–/–{psi}V–, AIDR{psi}V–, AIDR{psi}V–E2A+/–, AIDR{psi}V–E2A–/–, AIDR{psi} V–E2ARtE12, and AIDR{psi}V–E2ARtE47 (Fig. 3, A and B, and Table II). This analysis revealed a significant reduction in the percentages of sIg– cells for the heterozygous E2A mutant AIDR{psi}V–E2A+/– (8.1%) and the homozygous E2A mutant AIDR{psi}V–E2A–/– (5.5%) compared with the wild-type AIDR{psi}V– (19.7%) and the complemented E2A mutant clones AIDR{psi}V–E2ARtE12 (32.5%) and AIDR{psi}V–E2ARtE47 (29.9%). AID–/–{psi}V–, which has stopped all Ig hypermutation after the deletion of the AID cDNA expression cassette, was used as a control for a stable sIg+ clone. Only very few events (0.3%) accumulated in the sIg– gate of this clone.

Sequence analysis confirms that Ig hypermutation is reduced in the absence of E2A

To determine whether the lack of E2A changes the spectrum of hypermutations, the rearranged VJ regions of the L chain locus were sequenced from the AIDR{psi}V–E2A–/– clone 6 wk after subcloning (Fig. 4, A and C). This analysis confirmed that the AIDR{psi}V–E2A–/– clone accumulates mutations at a ~5-fold reduced rate compared with the AIDR{psi}V– progenitor clone. Nevertheless, the distribution of the mutations and the mutation spectrum of the AIDR{psi}V–E2A–/– clone was very similar to what was previously reported for the AIDR{psi}V– clone (18, 19). In addition, VJ regions of the complemented clones AIDR{psi}V–E2A RtE12 and AIDR{psi}V–E2ARtE47 were sequenced. Consistent with the FACS data, these clones had accumulated mutations at a rate similar to or higher than what was observed for the AIDR{psi}V– clone (Fig. 4, B and C). The analysis of the mutation spectrum of the complemented clones showed some deviations from the mutation spectrum of the AIDR{psi}V– progenitor clone, for example, a relative high percentage of T->A mutations in the AIDR{psi}V–E2ARtE12 and C->A mutations in the AIDR{psi}V–E2ARtE47 clones. However, the observed differences are not consistent between the two complemented clones and within different subclones of the same clone (data not shown) indicating that they reflect most likely fluctuation and different timing of mutations during the expansion of the sequenced subclones.


Figure 4
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 4. Comparison of mutations from AIDR{psi}V–E2A–/–, AIDR{psi}V–E2ARtE12, AIDR{psi}V–E2ARtE47, and AIDR{psi}V– cells. Mutations within the rearranged VJ L chain segments 6 wk after subcloning. The mutations of AIDR{psi}V– cells have been described previously (18 19 ). A, Single-nucleotide substitutions identified in the AIDR{psi}V–E2A–/– cells are mapped onto the likely sequence of the precursor cell for the subclone. Occasional deletions and insertions are indicated. Hot-spot motifs (RGYW and its complement WRCY) are highlighted in bold letters. B, Single-nucleotide substitutions identified in the AIDR{psi}V–E2ARtE12 and AIDR{psi}V–E2ARtE47 cells are shown above and below the likely sequence of the precursor cell for the subclone, respectively. Differences between the progenitor sequences of AIDR{psi}V–E2ARtE12 and AIDR{psi}V–E2ARtE47 are indicated in the line below the sequence. Occasional deletions and insertions are indicated. Hot-spot motifs (RGYW and its complement WRCY) are highlighted in bold letters. C, Analysis of the mutation pattern of single-nucleotide substitutions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report in this study that the inactivation of the E2A gene markedly reduces Ig hypermutation without affecting Ig expression levels. The reduction of Ig hypermutation in the absence of E2A was independent of the AID expression levels, which were maintained constant under a constitutive promoter. Expression of either the E12 or the E47 cDNA can complement the Ig hypermutation defect in E2A-disrupted cells to wild-type levels, indicating that the decreased hypermutation activity is indeed due to the lack of E2A expression. The result also shows that sequence differences among the two E2A splice variants are not critical for the effect. This is the first report that an Ig enhancer binding transcription factor influences Ig hypermutation independent of Ig transcription and AID expression levels.

Because Ig hypermutation is only initiated by AID-mediated cytidine deamination and depends on further processing of the resulting uracils by uracil DNA glycosylase (19, 21) and error-prone polymerases (22, 23), it cannot be ruled out that the reduction of hypermutation in E2A-negative cells is caused by effects downstream of AID. However, the absence of consistent changes in the mutation spectrum of the E2A-deficient or -complemented clones compared with the wild-type E2A progenitor clone argues against this possibility. Other DT40 mutants that interfere with the conversion of AID-induced uracils into hypermutations showed clear changes in the mutation spectrum like a transitions bias in uracil DNA glycosylase-deficient cells (19) and selectively reduced C-to-G and G-to-C mutations after REV1 disruption (Ref. 23 ; H. Arakawa, G.-L. Moldovan, H. Saribasak, N. N. Saribasak, S. Jentsch, and J.-M. Buerstedde, unpublished results).

In a more likely scenario, E2A influences Ig hypermutation upstream of AID action. This is consistent with the known role of the E2A-encoded proteins E12 and E47 as transcription factors which recognize E-box consensus motifs in the enhancers of their target genes. Because E-box sequence motifs are present in the enhancers of Ig and hypermutating non-Ig genes (13), it is tempting to speculate that E12 and E47 influence Ig hypermutation by binding to the Ig enhancers. This is also consistent with the observation that the introduction of an E-box sequence stimulated the mutation rate of a transgene (6). It remains unknown how the binding of the E2A encoded proteins to the Ig enhancers might stimulate Ig hypermutation, but the effect is apparently not due to the stimulation of Ig transcription activity, because no decrease of L chain transcription was observed in the E2A-negative mutant. One of the most intriguing possibilities would be that the E2A-encoded proteins either specifically recruit AID to the Ig loci or stimulate the AID cytidine deaminase activity. Studies of protein-protein interaction and detailed mutation analysis of the E2A coding regions should provide further insight into the mechanism responsible for the enhancement of Ig hypermutation by E2A.

AID expression in wild-type DT40 cells, which in contrast to the AIDR{psi}V– clone harbor pseudo-V gene conversion donors upstream of the rearranged L chain VJ segment, manifests itself by Ig gene conversion. If Ig hypermutation is enhanced by E2A at a step before AID-mediated cytosine deamination, one would expect that E2A expression will stimulate not only Ig hypermutation but also Ig gene conversion. This prediction has recently been confirmed by reporting increased gene conversion in DT40 cells that overexpress the E47 cDNA (24).


   Acknowledgments
 
We are grateful to Hiroshi Arakawa, Randy Caldwell, Huseyin Saribasak, and Jürgen Bachl for advice and critically reading the manuscript, and to Claire Brellinger, Martina Schreiber, and Benjamin Conci for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
J.-M. Buerstedde and U. Schoetz are inventors on a patent with the name "Method of Enhancing Gene Conversion, Somatic Hypermutation, and Class Switch Recombination." Also involved with the patent is the GSF-Forschungszentrum für Umwelt und Gesundheit, GmbH.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the European Framework VI Grant "Geninteg" and the Deutsche Forschungsgemeinschaft Sonderforschungsbereich "Networks in Genome Expression and Maintenance." Back

2 Current address: Dipartimento di Biologia, Università "Roma Tre," 00146 Rome, Italy. Back

3 Address correspondence and reprint requests to Dr. Jean-Marie Buerstedde, Institute of Molecular Radiobiology, GSF National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany. E-mail address: buersted{at}gsf.de Back

4 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; s, surface. Back

Received for publication January 20, 2006. Accepted for publication April 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Neuberger, M. S., R. S. Harris, J. Di Noia, S. K. Petersen-Mahrt. 2003. Immunity through DNA deamination. Trends Biochem. Sci. 28: 305-312. [Medline]
  2. Peters, A., U. Storb. 1996. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4: 57-65. [Medline]
  3. Gordon, M. S., C. M. Kanegai, J. R. Doerr, R. Wall. 2003. Somatic hypermutation of the B cell receptor genes B29 (Igbeta, CD79b) and mb1 (Ig{alpha}, CD79a). Proc. Natl. Acad. Sci. USA 100: 4126-4131. [Abstract/Free Full Text]
  4. Betz, A. G., C. Milstein, A. Gonzalez-Fernandez, R. Pannell, T. Larson, M. S. Neuberger. 1994. Elements regulating somatic hypermutation of an immunoglobulin {kappa} gene: critical role for the intron enhancer/matrix attachment region. Cell 77: 239-248. [Medline]
  5. Storb, U., A. Peters, E. Klotz, N. Kim, H. M. Shen, J. Hackett, B. Rogerson, T. E. Martin. 1998. cis-Acting sequences that affect somatic hypermutation of Ig genes. Immunol. Rev. 162: 153-160. [Medline]
  6. Michael, N., H. M. Shen, S. Longerich, N. Kim, A. Longacre, U. Storb. 2003. The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription. Immunity 1: 235-242.
  7. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553-563. [Medline]
  8. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565-575. [Medline]
  9. Arakawa, H., J. Hauschild, J. M. Buerstedde. 2002. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295: 1301-1306. [Abstract/Free Full Text]
  10. Harris, R. S., J. E. Sale, S. K. Petersen-Mahrt, M. S. Neuberger. 2002. AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr. Biol. 12: 435-438. [Medline]
  11. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, M. S. Neuberger. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12: 1748-1755. [Medline]
  12. Yoshikawa, K., I. M. Okazaki, T. Eto, K. Kinoshita, M. Muramatsu, H. Nagaoka, T. Honjo. 2002. AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 14: 2033-2036.
  13. Kotani, A., I. M. Okazaki, M. Muramatsu, K. Kinoshita, N. A. Begum, T. Nakajima, H. Saito, T. Honjo. 2005. A target selection of somatic hypermutations is regulated similarly between T and B cells upon activation-induced cytidine deaminase expression. Proc. Natl. Acad. Sci. USA 102: 4506-4511. [Abstract/Free Full Text]
  14. Kee, B. L., M. W. Quong, C. Murre. 2000. E2A proteins: essential regulators at multiple stages of B-cell development. Immunol. Rev. 175: 138-149. [Medline]
  15. Conlon, T. M., K. B. Meyer. 2004. Cloning and functional characterisation of avian transcription factor E2A. BMC Immunol. 14: 5-11.
  16. Arakawa, H., D. Lodygin, J. M. Buerstedde. 2001. Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnol. 1: 7[Medline]
  17. Caldwell, R. B., A. M. Kierzek, H. Arakawa, Y. Bezzubov, J. Zaim, P. Fiedler, S. Kutter, A. Blagodatski, D. Kostovska, M. Koter, et al 2005. Full-length cDNAs from chicken bursal lymphocytes to facilitate gene function analysis. Genome Biol. 6: R6[Medline]
  18. Arakawa, H., H. Saribasak, J. M. Buerstedde. 2004. Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. PLoS Biol. 2: 967-974.
  19. Saribasak, H., N. N. Saribasak, F. M. Ipek, J. W. Ellwart, H. Arakawa, J. M. Buerstedde. 2006. Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations. J. Immunol. 176: 365-371. [Abstract/Free Full Text]
  20. Angerer, W. P.. 2001. An explicit representation of the Luria-Delbrück distribution. J. Math. Biol. 42: 145-174. [Medline]
  21. Di Noia, J. M., M. S. Neuberger. 2002. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419: 43-48. [Medline]
  22. Simpson, L. J., J. E. Sale. 2003. REV1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line. EMBO J. 22: 1654-1664. [Medline]
  23. Ross, A. L., J. E. Sale. 2006. The catalytic activity of REV1 is employed during immunoglobulin gene diversification in DT40. Mol. Immunol. 43: 1587-1594. [Medline]
  24. Conlon, T., K. Meyer. 2006. The chicken Ig light chain 3'-enhancer is essential for gene expression and regulates gene conversion via the transcription factor E2A. Eur. J. Immunol. 36: 139-148. [Medline]



This article has been cited by other articles:


Home page
 Phil Trans R Soc BHome page
H. Arakawa and J.-M. Buerstedde
Activation-induced cytidine deaminase-mediated hypermutation in the DT40 cell line
Phil Trans R Soc B, March 12, 2009; 364(1517): 639 - 644.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Yabuki, E. C. Ordinario, W. J. Cummings, M. M. Fujii, and N. Maizels
E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification
J. Immunol., January 1, 2009; 182(1): 408 - 415.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Xiang and W. T. Garrard
The Downstream Transcriptional Enhancer, Ed, Positively Regulates Mouse Ig{kappa} Gene Expression and Somatic Hypermutation
J. Immunol., May 15, 2008; 180(10): 6725 - 6732.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Kothapalli, D. D. Norton, and S. D. Fugmann
Cutting Edge: A cis-Acting DNA Element Targets AID-Mediated Sequence Diversification to the Chicken Ig Light Chain Gene Locus
J. Immunol., February 15, 2008; 180(4): 2019 - 2023.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
H. Kitao, M. Kimura, K. Yamamoto, H. Seo, K. Namikoshi, Y. Agata, K. Ohta, and M. Takata
Regulation of histone H4 acetylation by transcription factor E2A in Ig gene conversion
Int. Immunol., February 1, 2008; 20(2): 277 - 284.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Ohm-Laursen and T. Barington
Analysis of 6912 Unselected Somatic Hypermutations in Human VDJ Rearrangements Reveals Lack of Strand Specificity and Correlation between Phase II Substitution Rates and Distance to the Nearest 3' Activation-Induced Cytidine Deaminase Target
J. Immunol., April 1, 2007; 178(7): 4322 - 4334.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
M. Nakayama, H. Suzuki, N. Yamamoto-Nagamatsu, H. K. Barman, H. Kikuchi, Y. Takami, K. Toyonaga, K. Yamashita, and T. Nakayama
HDAC2 controls IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions
Genes Cells, March 1, 2007; 12(3): 359 - 373.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. Y. Yang, S. D. Fugmann, and D. G. Schatz
Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences
J. Exp. Med., December 25, 2006; 203(13): 2919 - 2928.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schoetz, U.
Right arrow Articles by Buerstedde, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schoetz, U.
Right arrow Articles by Buerstedde, J.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS