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International Immunology Advance Access originally published online on January 7, 2008
International Immunology 2008 20(2):277-284; doi:10.1093/intimm/dxm140
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© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Regulation of histone H4 acetylation by transcription factor E2A in Ig gene conversion

Hiroyuki Kitao1,*, Masayo Kimura2,6,*, Kazuhiko Yamamoto2, Hidetaka Seo3, Keiko Namikoshi2, Yasutoshi Agata4, Kunihiro Ohta5 and Minoru Takata1

1 Department of Late Effect Studies, Radiation Biology Center, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
2 Department of Immunology and Molecular Genetics, Kawasaki Medical School, Okayama 701-0192, Japan
3 Chiome Bioscience Inc., Tokyo 113-0033, Japan
4 Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi-shi, Osaka 594-1101, Japan
5 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan
6 Present address: Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-5885, Japan

Correspondence to: M. Takata; E-mail: mtakata{at}house.rbc.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Recent studies implicate the transcription factor E2A in Ig diversification such as somatic hypermutation or gene conversion (GCV). GCV also requires active Ig transcription, expression of the activation-induced deaminase (AID) and a set of homologous recombination factors. We have disrupted the E2A gene in the chicken B-cell line DT40 and found greatly diminished rate of GCV without changes in the levels of transcripts from AID and Ig heavy chain or Ig light chain (IgL) genes. However, chromatin immunoprecipitation analysis revealed that the loss of E2A accompanies drastically reduced acetylation levels of the histone H4 in rearranged IgL locus. Furthermore, the defects in GCV were restored by trichostatin A treatment, which raised H4 acetylation to the normal levels. Thus, E2A may contribute to GCV by maintaining histone acetylation, which could be a prerequisite for targeting or full deaminase function of AID.

Keywords: activation-induced deaminase, chromatin immunoprecipitation, DT40 cell line


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
To fight against the overwhelming number of invading external organisms, adaptive immunity diversifies its repertoire of antigen-binding TCR or B-cell receptor by genetic mechanisms. In B cells, the rearranged V(D)J segments in Ig heavy chain (IgH) and Ig light chain (IgL) genes are further modified by single-nucleotide non-templated substitutions [somatic hypermutation (SHM)] (1) or by templated copying of nucleotide patches from upstream donor sequences [gene conversion (GCV)] (2, 3), leading to generation of antibody with higher affinity to antigen (4). Human or mouse utilizes the former mechanisms, while avian immune system exploits both mechanisms (1–3).

It has been firmly established that active Ig transcription is essential for both SHM and GCV (1–3). Therefore, they occur only in rearranged and transcriptionally active alleles. SHM and GCV are also dependent on expression of the activation-induced deaminase (AID), which is thought to deaminate deoxycytidine to deoxyuridine in actively transcribed single-stranded DNA regions (1–3). This is the common initiating DNA lesion shared by both processes (5). Then, the lesion is modified by uracil DNA glycosylase or mismatch repair proteins, followed by machineries of homologous recombination (HR) in GCV or by error-prone polymerases in SHM, leading to distinct sequence alterations (1–3).

Although over-expression of AID alone in fibroblasts is able to induce frequent point mutations in a transgene (6), SHM or GCV occurs mostly in limited segments of rearranged Ig alleles encoding antigen-binding variable region (1–3). How AID is targeted to Ig gene is currently unknown. Interestingly, recent studies indicated that Ig transcription is essential but not sufficient for robust SHM or GCV. For example, a heterologous promoter could not substitute function of Ig promoter in SHM as well as GCV in swapping experiments even though it can maintain equal levels of Ig transcription (7). Likewise, DNA methylation at mouse Ig{kappa} locus inhibits SHM without affecting transcription (8). Alteration of histone modifications/chromatin structure could constitute mechanistic basis for these phenomenon. Indeed, it has been reported that higher levels of histone acetylation is detected at the variable region undergoing SHM relative to the constant region (9) or the rearranged allele of IgL gene compared with the unrearranged allele (10). Furthermore, treatment with a histone deacetylase inhibitor trichostatin A (TSA) leads to elevated H4 acetylation as well as increased rate of GCV with constant AID levels (10). A more recent study reported that artificial tethering of heterochromatin protein 1 into the donor sequence region lowers histone acetylation levels as well as GCV rate without changes in Ig transcription (11). Collectively, these results are consistent with a notion that modifications of histones such as acetylation could be an important factor that regulates SHM and GCV.

Recent studies implicate B-cell transcription factor E2A in SHM and GCV (12, 13). A role of E2A gene for Ig SHM or GCV has been shown by a gene-targeting experiment (13) or an ectopic expression of E2A (12), respectively. E2A is a basic helix-loop-helix protein that binds to DNA motif called E-box and critically governs B-cell development and differentiation (14). To investigate how E2A contributes to regulation of GCV, we generated E2A–/– cell line by gene targeting in chicken B-cell line DT40. DT40 continues to diversify its rearranged variable gene predominantly by GCV (15) and undergoes targeted integration at extremely high rate (16). We confirmed significantly reduced rate of GCV in our E2A–/– cells without appreciable changes in mRNA levels of IgH and IgL genes or AID. Importantly, we found that acetylation levels of histone H4 were drastically reduced in rearranged IgL locus. Furthermore, the defects in Ig GCV were restored by ectopic expression of AID, as well as TSA treatment, which raised H4 acetylation to normal levels. We propose that E2A may contribute to Ig GCV by maintaining histone acetylation, which could be a prerequisite for AID to be targeted to the Ig locus or to mediate deaminase function.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell culture and transfection
DT40Cre1 wild-type cells and its derivatives were cultured in 39.5°C with 5% CO2 by using RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% chicken serum, 50 µM 2-mercaptoethanol and 2 mM L-glutamine. Transfection by electroporation and subsequent selection were done as described previously (17).

Construction of the E2A-targeting vectors and the E2A and AID cDNA expression vectors
A part of chicken E2A gene was amplified by long-range PCR using DT40 genomic DNA as template. The genomic PCR fragments were cloned into upstream and downstream of the floxed drug resistance marker cassettes, bsr and puro (18). The full-length cDNA for chicken E2A (E47) or AID was obtained by reverse transcription (RT)–PCR using Primescript® RT–PCR kit and Primestar® HS DNA polymerase (Takara Bio Inc., Ohtsu, Japan) from total RNA isolated from DT40 and inserted into the expression vector pcDNA3.1zeo-IRES-EGFP. This bicistronic vector was created by inserting the BglII–NotI fragment containing IRES-EGFP derived from pIRES2-EGFP (Clontech, Mountain View, CA, USA) into BamHI–NotI sites in pcDNA3.1zeo (Invitrogen, Carlsbad, CA, USA).

Generation of the E2A mutant clones
To generate the E2A mutant clones, we used the surface IgM (sIgM)-negative DT40 clone DT40Cre1, which has a defined frameshift mutation within the V{lambda}1 gene of rearranged allele that can be corrected by ongoing GCV at very high frequency (19, 20). We disrupted two alleles of E2A gene by sequential transfection using targeting vectors, E2A-bsr and E2A-puro. Target integration was screened by Southern blotting of EcoRI-digested genomic DNA. The disruption of E2A gene was further confirmed by amplifying full coding regions of E2A and Rad51 genes by RT–PCR.

Northern blot analysis
Northern blot analysis was done as described previously (19). The probes for chicken IgH Cµ and IgL C{lambda} were kindly provided by Masao Ono (Rikkyo University, Tokyo, Japan) (21).

Quantitation of mRNA by real-time RT–PCR
Total RNA was extracted and cDNA synthesized using Primescript® RT–PCR kit. Real-time PCR was done using Power SYBR Green Master Mix and 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative expression levels of E2A, PAX5, EBF and AID were determined by comparative CT method using GAPDH as an endogenous reference. To verify that contamination of chicken genome was negligible, each cDNA sample was compared with control amplification without RT. The following primer pairs were used to amplify 120-bp fragment from each chicken mRNA: E2A forward, 5'-GGGCCTTGCAGGCACAT-3' and E2A reverse, 5'-ATGGCTTCGTCCAACCTGTCT-3'; EBF1 forward, 5'-GGATCAGGACGGAACAGGATT-3' and EBF1 reverse, TTTCGTGCGTGAGCAGAACT-3'; PAX5 forward, 5'-CCACACCCAAAGTTGTCGAA-3' and PAX5 reverse, 5'-TGGGCACGGTGTCGTTATC-3'; AID forward, 5'-CTGCTACCGCATCACATGGT-3' and AID reverse, 5'-TAGAGGCGGGCAGTGAAAAT-3' and GAPDH forward, 5'-CCATCACAGCCACACAGAAGA-3' and GAPDH reverse, 5'-CTTTCCCCACAGCCTTAGCA-3'.

Measurement of GCV events at IgL locus
The rate of gaining sIgM expression was monitored by flow cytometric analysis of cells that had been kept in culture for indicated time after limiting dilution and then stained with FITC- or RPE-conjugated anti-chicken IgM (Bethyl, Inc., Montgomery, TX, USA). The percent sIgM-positive cells were calculated in live cell gate defined by propidium iodide staining and light-scatter profile. sIgM-positive cells were magnetic sorted by staining with anti-chicken IgM–FITC and anti-FITC magnetic beads (Miltenyi Biotech, Auburn, CA, USA). PCR amplification and sequencing of the rearranged V{lambda}1 gene was done as described previously (19). Nucleotide changes were aligned with the pseudogene database and then classified into GCV, non-templated point mutation (SHM) or ambiguous category according to the criteria described (5). Statistical analysis was done by using StatView software (SAS Institute, Inc., Cary, NC, USA).

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) was done as described previously (10). Briefly, cells were fixed with formaldehyde, extracted with lysis buffer [1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, 50 mM HEPES–KOH (pH 7.5) and 150 mM NaCl] and sonicated. Immunoprecipitation was done using anti-acetylated histsone H4 (Upstate Biotechnology, Lake Placid, NY, USA) and Dynabeads-protein A (Invitrogen). Precipitated chromatin was extensively washed, reversed by heat treatment and DNA fragments were purified by phenol extraction. Primers to amplify rearranged (IGLC503-IGLC2995) and unrearranged (IGLC503–IGLC992) allele of IgL gene locus and ovalbumin promoter region (OVA419–OVA923) were described elsewhere (10).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Targeted disruption of E2A gene in DT40Cre1 cells
To study the roles of E2A in GCV, we disrupted the E2A gene in a DT40 subline, DT40Cre1, by gene targeting. Since DT40Cre1 cells do not express sIgM because of a frameshift mutation in the complementarity-determining region 1 (CDR1) of IgL V{lambda}1 gene, which can be corrected by GCV at very high rate (20), we decided to use this cell line. DT40Cre1 cells were serially transfected with targeting vectors (Fig. 1A), and clones carrying a targeted event were identified by Southern blotting (Fig. 1B). Both of the two isoforms E12 and E47 encoded by the E2A gene were expected to be disrupted since the gene targeting was designed to replace the genomic region containing exons shared by these two isoforms with the resistance gene cassettes. We confirmed the loss of intact E2A mRNA by RT–PCR using primers that amplify full coding region (Fig. 1C) as well as real-time PCR (Fig. 1F).


Figure 1
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  		Fig. 1. Generation and characterization of E2A-deficient DT40 cells. (A) A scheme of E2A gene targeting. The location of disrupted exons and E47-specific exon are indicated by black boxes. Triangles indicate loxP sites which floxed the resistance gene cassettes bsr or puro. Cre-mediated excision of the cassettes was not done in this study. E, EcoRI and B, BamHI. (B) Southern blot analysis of the EcoRI-digested genome DNA from cells with indicated genotypes. (C) mRNA levels of E2A, EBF, PAX5 and Rad51 in cells with indicated genotypes as revealed by RT–PCR analysis. WT, wild type. (D) Northern blot analysis of IgH Cµ, IgL C{lambda} and AID mRNA expression levels. Amount of 28S ribosomal RNA is shown as loading control for northern gel electrophoresis. (E) Semi-quantitative RT–PCR analysis of IgH Cµ, IgL C{lambda} and AID mRNA. cDNAs prepared from the cells were serially diluted and subjected to appropriate cycles of PCR amplification. GAPDH was also amplified as control. N, PCR without cDNA template. (F) Real-time PCR analysis of E2A, EBF, PAX5 or AID expression. cDNAs were prepared as in (E) and real-time PCR was performed using SYBR green. Expression levels were calculated as relative quantity to those in wild-type cells. The amplification was done in triplicate and repeated three times. Mean and standard deviation of the representative data set are shown.

 
A hierarchy as well as feedback mechanisms are reported among B-cell transcription factors. E2A and EBF synergistically activate B lymphopoiesis, which may be due to regulation of the essential B-lineage commitment factor PAX5 (22). Thus, we looked at mRNA levels of PAX5 and EBF in E2A–/– clones (termed E2A–/– #1 and E2A–/– #2) by RT–PCR (Fig. 1C) and real-time RT–PCR (Fig. 1F). The RT–PCR analysis showed that the levels of EBF transcripts were not affected, while mRNA levels of PAX5 gene appeared to be mildly decreased, and this was confirmed by the real-time PCR. The growth rate of two independent clones of E2A–/– (E2A–/– #1 and E2A–/– #2) was the same as that of wild-type cells (Fig. 2A). Consistent with this, no clear difference was observed in their cell-cycle profile (data not shown).


Figure 2
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  		Fig. 2. Characterization of E2A–/– clones. (A) Growth curve of the cells with indicated genotypes. Cell numbers were counted using flow cytometer with plastic microbeads as standards. (B) sIgM expression in cells before and after expansion for 3 weeks. Examples of FACS profiles are shown in each genotype. (C) Fluctuation analysis of sIgM expression. Clonal sub-populations (n = 24) with indicated genotypes were expanded for 3 weeks and examined using FACSCalibur. Mean and standard deviation of percentage sIgM-positive cells are shown. (D) GCV and SHM events in wild-type and E2A–/– cells. Horizontal thick lines indicate GCV tracts; lollipop stands for SHM events; vertical lines indicate ambiguous single-nucleotide changes. Triangles indicate location of the original frameshift in IgL V{lambda}1 gene. Identified pseudogene donors that corrected the original frameshift are also shown (e.g. {Psi}V8).

 
Decreased rate of GCV in E2A–/– clones
To see whether disruption of E2A can affect Ig GCV, we carried out fluctuation analysis using DT40Cre1 wild-type and E2A–/– clones. Cells were sub-cloned by limiting dilution, propagated for 3 weeks and 24 or more clonal sub-populations were analyzed for their sIgM expression (Fig. 2B and C). In wild-type sub-populations, 34.3% of cells became sIgM positive in average. On the other hand, only 11.3% (E2A–/– #1) or 12.6% (E2A–/– #2) of cells were converted to sIgM positive in E2A–/– clones (Fig. 2C). The difference in percentage sIgM-positive cells between wild type and E2A–/– #1 or E2A–/– #2 was statistically significant (P < 0.0001, Mann–Whitney U analysis).

To confirm that the emergence of sIgM-positive cells is the result of GCV in V{lambda}1 gene, we purified sIgM-positive cells in wild-type and E2A–/– sub-populations using anti-chicken IgM antibody and magnetic beads. Then, the rearranged V{lambda}1 gene was amplified from their genomic DNA, cloned into plasmid vector and directly sequenced. In both genotypes, the original frameshift mutation in CDR1 of the V{lambda}1 gene was corrected in all sequences as a result of GCV (Fig. 2D). The effect of the E2A deficiency was probably not limited to the GCV involving CDR1 since the number of GCV events outside of the CDR1 was also decreased (15 events in 36 sequences from wild-type cells versus 7 events in 40 sequences from E2A–/– #1 cells) (Fig. 2D). The usage of pseudogene templates appeared not to be drastically altered (Fig. 2D), although mean number of nucleotides that have been changed from original sequence (tract length) was slightly but significantly longer in E2A–/– cells (20.4 versus 29.1, P = 0.0271, Mann–Whitney U analysis). Since the cell growth of wild type and E2A–/– are indistinguishable (Fig. 2A), we conclude that efficiency of GCV per single cell cycle is reduced in E2A–/– cells.

Decreased histone H4 acetylation at rearranged IgL allele in E2A–/– cells
It has been well established that GCV requires active transcription of Ig gene and expression of the key factor AID (2, 20, 23). Since E2A activates transcription of AID as well as Ig genes (14, 22, 24, 25), we first hypothesized that the expression levels of AID or Ig gene itself may be reduced. However, we could not detect any significant decrease in the transcript levels of AID and IgL C{lambda} or IgH Cµ genes in northern blot analysis (Fig. 1D) and semi-quantitative RT–PCR (Fig. 1E). Real-time RT–PCR also confirmed that AID expression was not altered (Fig. 1F).

The process that affects nucleosomal DNA such as GCV or SHM might be regulated by histone modifications. It has been reported that E2A physically associates with histone acetyltransferases (HATs), p300, CBP and PCAF (26). Thus, we hypothesized that E2A may modulate histone acetylation by recruiting HATs to rearranged Ig loci, and the acetylation levels at rearranged IgL gene may be decreased in the absence of E2A. To test this possibility, we performed ChIP analysis at IgL gene locus using anti-acetylated histone H4 antibody in wild-type and E2A–/– cells. Consistent with the previous report (10), high levels of histone H4 acetylation were observed at the rearranged allele of IgL gene in wild-type cells compared with the levels at the unrearranged allele (Fig. 3B, lane 4). Importantly, in E2A–/– genetic background, levels of histone H4 acetylation were significantly reduced in rearranged allele of IgL gene (Fig. 3B, lanes 5 and 6). The reduced acetylation levels were restored in E47-transfected E2A–/– #1 cells, indicating that the observed acetylation defects were indeed caused by loss of E2A (Fig. 3C). Thus, E2A deficiency accompanied reduced levels of histone H4 acetylation, which seemed to correlate with defects in Ig GCV. These results also indicated that the reduced levels of H4 acetylation were sufficient to maintain transcript levels of Ig gene in E2A–/– cells.


Figure 3
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  		Fig. 3. ChIP analysis of IgL locus. (A) Position of the primers to specifically detect rearranged or unrearranged IgL V{lambda} gene locus. (B) ChIP analysis. WT, wild-type and #1 or #2, E2A–/– #1 or E2A–/– #2, respectively. Fixed chromatin of the cells was solubilized and subjected to immunoprecipitation with or without anti-acetylated histone H4 antibody ({alpha}-AcH4). Part of IgL locus was amplified as indicated in (A) from solubilized lysate (input) or precipitates (IP). Ovalbumin promoter was analyzed as a control. The experiment was repeated at least three times with similar results. (C) ChIP analysis in E47-complemented cells. E2A–/– #1 cells were transfected with expression vector pcDNA3.1zeo/chE2A(E47)-IRES-EGFP and EGFP expression was analyzed with FACSCalibur (upper panel). Reconstituted cells (R) were subjected to ChIP analysis as in (B) (lower panel).

 
TSA treatment can bypass the E2A requirement in Ig GCV as well as in histone H4 acetylation
Next, we wished to examine effects of TSA treatment in E2A–/– cells. First, we cultured E2A–/– cells in the presence or absence of 2.5 ng ml–1 TSA for 6 h and measured the acetylation levels of histone H4 at rearranged allele of IgL gene by ChIP analysis. The H4 acetylation levels in E2A–/– cells were enhanced by TSA treatment to the levels similar to those in wild-type DT40Cre1 cells (Fig. 4A). The acetylation levels in wild-type cells were not enhanced further in the presence of TSA for 6 h (Fig. 4A); however, this could be increased by more prolonged incubation as described (10).


Figure 4
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  		Fig. 4. Histone H4 acetylation and GCV in cells treated with TSA. (A) Levels of H4 acetylation of rearranged IgL locus. Cells were treated with or without TSA for 6 h. ChIP analysis was performed as described in Fig. 3(B) legend. The experiment was repeated at least three times with similar results. (B) Fluctuation analysis of sIgM expression. Clonal sub-populations (n = 12) of E2A–/– clones were kept in culture for 3 weeks with or without TSA and were analyzed using FACSCalibur. Mean and standard deviation of percentage sIgM-positive cells are shown. (C) Semi-quantitative RT–PCR analysis of IgH Cµ, IgL C{lambda} and AID mRNA. cDNAs were prepared from the cells with or without treatment with TSA (2.5 ng ml–1 for 6 h) and analyzed as in Fig. 1(E). N, PCR without cDNA template.

 
Then, we looked at whether TSA has any effect on GCV rate by fluctuation assay. Two independent E2A–/– cell lines were each cloned by limiting dilution, and 12 sub-populations were kept in culture for 3 weeks in the presence or absence of 2.5 ng ml–1 TSA. Strikingly, 78.4% (E2A–/– #1) and 69.9% (E2A–/– #2) of cells converted to sIgM positive in average when we continuously added TSA during culture period. In contrast, only 10.1% (E2A–/– #1) and 11.4% (E2A–/– #2) of cells in each sub-population expressed sIgM in the absence of TSA (Fig. 4B). The mRNA expression levels of IgL C{lambda}, IgH Cµ and AID genes did not change by culturing with 2.5 ng ml–1 TSA for 6 h either in wild-type or in E2A–/– #1 cells (Fig. 4C). Thus, TSA treatment can bypass the requirement for E2A in the acetylation of histone H4 as well as efficient GCV. However, this effect may not be mediated by enhancing the expression of AID, IgL and IgH genes.

Enforced expression of AID can partially reverse the defective Ig GCV in E2A–/– cells
TSA treatment dramatically enhanced histone H4 acetylation and GCV at rearranged allele of IgL gene in E2A–/– genetic background. Histone H4 acetylation may indicate more open chromatin structure with better accessibility of molecules to DNA in chromatin. We hypothesized that accessibility of AID to Ig loci may be impaired in E2A–/– cells and over-expression of AID might overcome decreased chromatin accessibility. To examine whether AID expression level has an impact on GCV frequency, we established stable E2A–/– cell lines that express chicken AID gene together with EGFP in the bicistronic manner.

GFP expression in transfected E2A–/– cells was confirmed by flow cytometric analysis (Fig. 5A). Using this cell clone, we carried out fluctuation analysis as before except for the shorter culture period (2 weeks). In clonal sub-populations derived from original E2A–/– cells, 2.2% of cells in average became sIgM positive. On the other hand, in sub-clones from E2A–/– cells expressing AID–IRES–EGFP, 4.5% of cells turned to be sIgM positive, confirming that, albeit weakly, exogenous AID expression enhances Ig GCV (Fig. 5B; P < 0.0001, Mann–Whitney U analysis).


Figure 5
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  		Fig. 5. Effect of ectopic AID expression on GCV rate. (A) Expression of chicken AID (chAID) in E2A–/– cells. E2A–/– #1 cells were transfected with expression vector pcDNA3.1zeo/chAID-IRES-EGFP, and EGFP expression was analyzed with FACSCalibur. Cells without transfection were also analyzed as control. (B) Fluctuation analysis of sIgM expression. Clonal sub-populations (n = 24) of E2A–/– #1 cells with or without transfected chAID were kept in culture for 2 weeks and were analyzed using FACSCalibur. Mean and standard deviation of percentage sIgM-positive cells are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we confirmed a crucial role of E2A gene for GCV using gene-disrupted DT40Cre1 cells. GCV is a complex process that requires (i) AID that initiates DNA lesions which are further modified by other factors, (ii) active transcription at rearranged Ig gene and (iii) DNA repair by HR (2, 3). Since E2A is a critical transcription factor for B-cell differentiation (14, 22), we first postulated lowered expression of Ig or AID gene as an explanation for the defective GCV. However, mRNA levels of IgL, IgH and AID genes were not appreciably decreased in our E2A–/– cells. In addition, expression levels of sIgM in converted E2A–/– cells were comparable to those in wild-type DT40Cre1 cells (Fig. 2B). Consistently, Buerstedde et al. reported that inactivation of E2A gene in an engineered DT40Cre1 subline (termed AIDR{Psi}V–) reduces Ig hypermutation without affecting Ig expression levels (13). Similarly, expression of the E2A isoform E47 increased Ig GCV without elevating Ig transcripts (12). On the contrary, Nakayama et al. (27) reported decreased levels of Ig transcripts in their E2A–/– DT40 mutants. This discrepancy could be due to use of different DT40 sub-clone as parental cells (DT40Cre1 versus original DT40).

In addition to these observations, recent evidence that histone H4 acetylation has crucial roles for GCV (10) prompted us to examine the acetylation levels in E2A–/– cells. Our ChIP analysis clearly showed that the loss of E2A was associated with significantly decreased levels of histone H4 acetylation in rearranged IgL allele. Furthermore, TSA treatment reversed GCV defects as well as diminished H4 acetylation. Histone acetylation has been proposed to contribute to chromatin accessibility (28). Since E2A associates with HATs (26) and cis-elements in Ig gene are important for Ig diversification (1), it is tempting to speculate that E2A may recruit HATs to Ig loci by binding to cis-elements containing E2A-binding E-box motifs. Although a recent gene-targeting experiment in DT40 revealed that the IgL enhancer containing E-box is dispensable for GCV (7), there are several E-box motifs around chicken IgL V{lambda}1 in the database, which may serve as additional binding sites for E2A.

Thus, these data are consistent with the hypothesis that lowered levels of histone H4 acetylation in E2A–/– cells may limit the access of AID to target DNA in chromatin of Ig gene. The limited access is not absolute because E2A–/– cells displayed some GCV events, which could be enhanced by TSA treatment. This suggests that there might be an additional factor that weakly recruits AID by itself, which requires histone acetylation for full function. It is also possible that the hyperacetylation allows AID to catalyze deamination reaction more efficiently. Alternatively, hyperacetylation in chromatin may enhance processes downstream of AID such as HR reaction. The fact that average tract length was slightly longer in E2A–/– cells may support the view that AID-initiated HR reaction itself was also affected in the absence of E2A. It has been suggested that histone acetylation levels affect loading of DNA repair proteins at DNA break ends (29). Longer tract length may indicate that less open chromatin structure could modify later steps in GCV such as processing of recombination intermediate (30). These possibilities are not mutually exclusive and should be clarified in future studies.

In summary, using E2A-disrupted cells and TSA treatment, we found good correlation between histone H4 acetylation and efficiency in GCV. AID has to be targeted to rearranged Ig loci to avoid catastrophic effects of genomic instability on other genes. E2A may contribute to Ig GCV by maintaining histone acetylation, which could be a prerequisite for AID to be targeted to the Ig locus or to exert full deaminase function.


   Acknowledgements
 
We would like to thank Masao Ono (Rikkyo University) for providing chicken Ig probes; Hiroshi Arakawa and Jean-Marie Buerstedde (GSF Institute, Munich, Germany) for providing DT40Cre1 cells and the floxed puro resistance gene cassette; Toshiyuki Habu and Junya Kobayashi (Radiation Biology Center, Kyoto University) for extensive help in real-time PCR experiments and Munehisa Yabuki and W. Jason Cummings (Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA) and Kazuo Kinoshita (Evolutionary Medicine, Shiga Medical Center Research Institute, Shiga, Japan) for advice and critical reading of the manuscript.


   Abbreviations
 
AID, activation-induced deaminase
ChIP, chromatin immunoprecipitation
CDR1, complementarity determining region 1
GCV, gene conversion
HAT, histone acetyltransferase
HR, homologous recombination
IgH, Ig heavy chain
IgL, Ig light chain
RT, reverse transcription
SHM, somatic hypermutation
sIgM, surface IgM
TSA, trichostatin A

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: T. Kurosaki

Received 5 September 2007, accepted 30 November 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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