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1 Shibata Distinguished Scientist Laboratory, RIKEN Discovery Research Institute, and 2 Chiome Bioscience Inc., RIKEN, 2-1 Hirosawa, Wako-shi, Saitama-ken 351-0198, Japan
3 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
4 REDS Group, JST, Saitama Small Enterprise Promotion Corporation, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, Japan
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Abstract |
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Introduction |
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Modification of chromatin structures is an important factor of regulation for DNA-related processes in eukaryotes, including transcription and recombination. Notably, reversible acetylation of nucleosome histones is believed to play a significant role in the regulation of gene expression, by inducing the local opening of chromatin structures and providing recognition sites for transcriptional activators containing bromodomains (Shahbazian & Grunstein 2007). Previous works also have shown that homologous recombination during meiosis is accompanied by the hyperacetylation of histones around the recombination hotspots (Yamada et al. 2004). Regarding Ig diversification, chromatin acetylation has also been reported to promote V(D)J recombination (Kwon et al. 2000; McBlane & Boyes 2000; McMurry & Krangel 2000) and class switch recombination (Nambu et al. 2003; Li et al. 2004), presumably by facilitating the accessibility of recombination factors to DNA. Histone hyperacetylation would be therefore expected to enhance Ig gene conversion as well.
Based on these observations, we have demonstrated in our previous report (Seo et al. 2005) that Ig gene conversion frequency in the chicken B cell line DT40 increases markedly upon treatment with trichostatin A (TSA), which induces an accumulation of acetylated histones in vivo by reversibly inhibiting histone deacetylase (HDAC) catalytic activity (Yoshida et al. 1990). The HDAC family comprises a dozen identified members, which are divided into four classes based on their sequence similarities (reviewed in Gallinari et al. 2007). As TSA induces a global increase of histone acetylation throughout the genome and affects most if not all members of the Rpd3-related class I and Hda1-related class II HDACs (Furumai et al. 2002), it is still unclear whether the stimulation of Ig gene conversion is caused directly by the enhancement of local chromatin accessibility at the Ig V region, or by some other effects such as the transcriptional regulation of recombination activators. Moreover, as an increasing number of studies point out that individual HDACs of a same class can play distinct regulatory roles (Nakayama & Takami 2001; Zupkovitz et al. 2006; Senese et al. 2007), the question arises to know whether one or several HDACs could be involved specifically in the control of Ig diversification. Most notably, other studies of DT40 mutants indicated that HDAC2 controls the expression of IgH (Takami et al. 1999) and IgL (Takechi et al. 2002), while in contrast the deletion of HDAC1 showed little effect (Takami et al. 1999) and HDAC3 was found essential to the viability of DT40 cells (Takami & Nakayama 2000).
We report here our analysis of the homozygous HDAC2–/– knockout DT40 mutant, showing that HDAC2 deletion is sufficient to increase the Ig gene conversion frequency at a much higher level than in wild-type (WT) cells. Remarkably, the distribution of gene conversion tracts observed along the Ig V region in the HDAC2–/– mutants was significantly different from those of TSA-treated WT cultures. Furthermore, the effects of the HDAC2 deletion and of the treatment with TSA were found to be additive. Our results suggest that the control of Ig gene conversion is the subject of subtle regulations involving distinct HDACs beyond their function in global histone acetylation and Ig transcription.
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Results |
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To analyze the frequency of gene conversion in a HDAC2-deficient context, we generated a HDAC2–/– deletion mutant in CL18, a surface IgM negative (sIgM–) DT40 subclone containing a frameshift mutation in the IgL variable (IgLV) locus (Buerstedde et al. 1990). After transfection with the knockout construct (Fig. 1A), stable transformants were identified by Southern blotting (data not shown) and absence of HDAC2 expression was confirmed by RT-PCR and Western blot analysis (Fig. 1B). We also confirmed that, as previously reported by Takami et al. (1999), the growth rates of the homozygous HDAC2–/– mutants were identical to that of the WT CL18 cells (Fig. 1C). To compare the effects of the HDAC2 deficiency to those induced by the inhibition of HDACs by TSA, we subcultured in parallel WT and HDAC2–/– cells in media supplemented with 5 or 10 nM TSA. Growth rates were noticeably decreased for the TSA-treated cultures depending of its concentration (Fig. 1C), and the HDAC2–/– mutant exhibited a much greater sensitivity to TSA than WT cells.
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Comparison of the effects of HDAC2 deletion and of TSA treatment on local histone acetylation at the IgL locus and on gene transcription
Our previous studies (Seo et al. 2005) showed that the treatment of DT40 cells with TSA enhances histone H4 acetylation locally in the Ig V region, which is accompanied by an induction of transcriptional activity at the recombination active IgL allele. Besides, HDAC2 deficiency is known to increase IgL and IgH transcription (Takami et al. 1999, 2002) concomitantly to the increase of bulk acetylation levels of core histones (Nakayama et al. 2007). To determine whether deletion of HDAC2 specifically enhances histone acetylation at the active IgL variable locus as well, we performed a chromatin immunoprecipitation assay (ChIP) with an antibody recognizing acetylated lysines 5, 8, 12 and 16 in the histone H4 N-terminus tail (Fig. 3). As previously observed (Seo et al. 2005), the WT histone H4 acetylation level was higher at the recombination active IgL allele than the inactive allele, and was increased by several folds at both alleles upon treatment with TSA depending on its concentration. As expected, enhancement of acetylation was also observed at a moderate level in the HDAC2–/– cells at both allele (two- to fourfold increase). Addition of 5 nM TSA in HDAC2–/– cultures also induced higher levels of acetylation in an additive manner, indicating that other HDACs might contribute to control H4 acetylation besides HDAC2.
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Comparative analysis of the distribution of sequence alterations along the IgL and IgH variable (V) regions in HDAC2–/– and TSA-treated DT40 cells
To better assess the specific features of the sequence alterations induced along the IgLV and IgHV regions, we cloned and sequenced the VJ-rearranged segments from WT and HDAC2–/– cells treated or not with TSA. Figure 5A illustrates the representative sequence patterns obtained for the active IgLV allele of HDAC2–/– and of TSA-treated WT cells after 2 months of culture. As expected from our previous results (Seo et al. 2005), most of the sequences of the TSA-treated WT culture (38 out of 42 randomly analyzed clones) displayed differences from the initial sequence prior to diversification. A striking observation was that the large majority of the HDAC2–/– sequences (39 out of 42) harbored multiple alterations as well, despite the lower level of reversion to sIgM+ detected by the aforementioned FACS analysis (Fig. 2B). In both TSA-treated WT and HDAC2–/– cultures, most of the changes could be attributed to gene conversion events (Fig. 5A). A few patterns also contained point mutations (around 20%) and deletion/insertion events (around 10%), but no significant difference was observed in their proportion between the two cultures. In contrast, we confirmed that alterations were very limited in the mock-treated WT control (not shown) and the heterozygous HDAC2+/– (30%, Fig. 6), and that no conversion event could be observed at the inactive allele (Supplementary Fig. S2R).
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From the above observations, we inferred that gene conversion in TSA-treated WT occurs preferentially around the CL18 mutation in CDR1, whereas they are more broadly distributed along the entire IgLV region in HDAC2–/–. This result was reliably reproduced in several independent experiments summarized in Fig. 6. For clearer comparison, we classified the IgLV patterns into four categories according to whether they displayed sequence alterations: (i) in CDR1 (meaning with mutations or conversion tracts starting within or upstream of CDR1), (ii) in regions downstream of CDR1 (with tracts non-overlapping CDR1), (iii) in both regions, or (iv) in neither region. Thus, we visualized a pronounced bias towards accumulation of alterations in CDR1 for the TSA-treated WT cells: after 1 month of culture, most of the patterns (91%) displayed sequence alterations, but the majority of them were localized exclusively within CDR1 (59%) and extremely few (1%) in regions downstream of CDR1. Inversely, in HDAC2–/– cells, although the total proportion of altered sequences was slightly lower (76%), the majority (30%) contained alterations downstream of CDR1, while 18% where found within CDR1. The HDAC2–/– cultures continued to differentiate broader diversification patterns during their expansion, so that after 2 months of culture, the total proportion of altered sequences increased further (85%), among which 32% were localized downstream of CDR1 and 15% within CDR1.
We next analyzed the IgLV sequences in HDAC2–/– cells treated with 5 nM TSA (Fig. 6, last chart; complete sequence patterns are indicated in Supplementary Fig. S2K–L). Almost all of the sequences (94%) showed diversified patterns after 1 month of culture. The proportion of sequences harboring two or more alterations attributable to independent events doubled (50/66, 76%) compared to TSA-treated WT and non-treated HDAC2–/– (respectively, 37% and 38%). Treatment with TSA enhanced gene conversion in CDR1, so that the large majority of the sequences (63%) were classified in category 3: with conversion tracts both in CDR1 and downstream of CDR1. Therefore, we conclude that the effects of HDAC2–/– and of treatment with TSA are also additive with respect to the spatial targeting of sequence alterations. Along with the conversion events, it should be noted that the proportion of aberrant deletions and insertions also appeared to increase additively, reaching 26% in average in TSA-treated HDAC2–/– cultures.
Finally, we also analyzed the sequence patterns of the IgHV region in HDAC2–/– and TSA-treated WT cultures (Figs 5B and 7; complete sequence patterns in Supplementary Fig. S2M–Q). Remarkably, IgH diversification appeared less active than for the IgL in TSA-treated WT, but was strongly enhanced in HDAC2–/–. While we could not find any obvious difference in the distribution patterns of alterations in the IgHV region, the kinetics of diversification increased strikingly faster in HDAC2–/– (84% of the sequences analyzed after 2 months of culture) than in TSA-treated WT (36%) (Fig. 7). Thus, HDAC2–/– cells were demonstrated to be more efficient in terms of Ig gene diversification at both IgLV and IgHV loci.
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Discussion |
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To ensure that the distinct distribution patterns of sequence alterations observed in HDAC2–/– and TSA-treated WT cultures were not due to any artifact caused by the selection of individual clones, we showed that they were reproducible using in each case at least four independent clonal cultures harvested at different times (Fig. 6 and Supplementary Fig. S2). We additionally noted that the preferential localization of gene conversion to CDR1 in TSA-treated cultures was also observed in the results of our previous publication (Seo et al. 2005) and in many unpublished experiments by ourselves. Since the sIgM– frameshift mutation of CL18 is located in CDR1, the preferential targeting of gene conversion towards CDR1 in TSA-treated cultures would explain the very high level of their reversion to sIgM+. Another possibility could be that in TSA-treated cultures, a vital advantage of sIgM+ over sIgM– cells would allow a selective expansion of the former, thereby leading to an enrichment of sequence alterations within IgL CDR1. However, this seems unlikely because first, the proliferation rate of sIgM+ and sIgM– cells appeared similar when analyzed by CFSE dye dilution assay (Supplementary Fig. S3). Second, we also observed that one of our clones did not express sIgM because of an independent frameshift mutation at the 5'-end of the V region (Supplementary Fig. S2–L), and in spite of it remaining in majority sIgM–, it still accumulated gene conversion tracts in CDR1 upon TSA treatment. Thus, we propose that the distinct distribution patterns of sequence alterations directly reflect differences in the preferential targeting of gene conversion.
It remains to be elucidated what kind of HDAC-related mechanisms are involved in the control of gene conversion. Histone acetylation has been shown to be associated with the activation of meiotic homologous recombination (Yamada et al. 2004), V(D)J recombination (Kwon et al. 2000; McBlane & Boyes 2000; McMurry & Krangel 2000), Ig class switch recombination (Nambu et al. 2003; Li et al. 2004) and Ig somatic hypermutation (Woo et al. 2003), although some contradicting results have been reported for the latter (Odegard et al. 2005). Deletion of HDAC2, as well as treatment with TSA, induced significant increases of the histone H4 acetylation levels around the active IgLV region accompanying the enhancement of gene conversion (Seo et al. 2005 and present report). Preliminary results also indicated some increase of acetylation levels at IgHV (data not shown). Thus, histone acetylation at the Ig locus might directly stimulate gene conversion by increasing local DNA accessibility in the V regions to facilitate the recruitment of recombination initiators or activators. On the other hand, HDAC2–/– and TSA-treated cells exhibited clearly distinct and additive responses in the targeting preferences of gene conversion, which would not be explained by a simple increase in the global level of acetylation in IgLV. Additional work would be required to assess subtle changes in chromatin configuration at a higher resolution or in distinct histone acetylation code determined by the differential contributions of individual HDACs and masked by the global acetylation level. Indeed, the effects are most likely different between the selective removal of HDAC2, the other members of the HDAC families remaining fully active, and the treatment with TSA, which inhibits many members of the HDAC families in a non-selective and partial manner.
Another possibility is that higher expression of Ig gene transcripts may result in the targeting and activation of gene conversion, since such mechanism has been proposed to explain the targeted hypermutation of IgV segments in mammalian cells (Bachl et al. 2001; Yoshikawa et al. 2002). In agreement with previous reports (Takami et al. 1999; Nakayama et al. 2007), we showed that IgL and IgH transcripts accumulated at a significantly higher level in HDAC2–/– compared to WT cells (Fig. 4). However, the treatment of WT and HDAC2–/– cells with TSA synergistically induced a much higher level of IgL and IgH transcripts in a concentration-depending manner. Therefore, while we cannot exclude a specific control of Ig gene expression by HDAC2 as it has been proposed (Nakayama et al. 2007), the stimulation of Ig expression seems more likely a consequence of the increase in histone acetylation levels and the resulting chromatin alteration at its promoter. All these results confirm the positive correlation between Ig diversification, histone acetylation and Ig transcription. However, the activated transcription per se is not sufficient to account for the distinct distribution patterns of targeted Ig gene conversion, nor for the elevated frequency of conversion at the IgH locus in HDAC2–/– cultures, since they displayed a lower Ig transcript levels than in TSA-treated WT. In this regard, recent reports of promoter substitution experiments (Yang et al. 2006) and of the transient targeting of AID-dependent mutations to newly integrated non-Ig DNA cassettes (Yang et al. 2007) demonstrated that active transcription alone is not necessarily sufficient to enhance Ig gene diversification, and suggested that Ig diversification could be modulated by other putative unidentified cis-acting regulators and by specific local properties of chromatin structure. We can therefore speculate that a combination of multiple transcription dependent and independent processes, the latter possibly involving modification of chromatin structures by HDAC2, allows the finely controlled targeting and activation of Ig gene conversion.
Several lines of evidence support that AID targets single-strand DNA (ssDNA) transiently exposed during transcription elongation to initiate hypermutation and recombination mechanisms (Bransteitter et al. 2003; Chaudhuri et al. 2003; Ramiro et al. 2003; Shen & Storb 2004; Besmer et al. 2006). Previous findings suggested in addition that formation of transcription-dependent R-loops in class switch regions (Yu et al. 2003) and of patches of chromatin-stabilized ssDNA in hypermutating regions (Ronai et al. 2007) may facilitate access of AID to its target substrates. Interestingly, it has been recently shown that hypermutation and recombination were strongly enhanced when expressing AID in yeast mutants deficient for the THO complex (Gomez-Gonzalez & Aguilera 2007), in which defect of co-transcriptional messenger ribonucleoprotein assembly promotes genomic instability and R-loop formation. Thus, an interesting hypothesis would be that the action of AID is stimulated in a context facilitating prolonged exposure of transcription-generated ssDNA. Chromatin modifications are tightly involved in each steps of progression into transcription (reviewed in Li et al. 2007) and notably, failure in histone deacetylation and redeposition at the end of elongation was proposed to enhance spurious transcription from cryptic promoters in yeast, presumably by leaving free DNA exposed to transcription factors (Carrozza et al. 2005; Joshi & Struhl 2005). It is therefore tempting to speculate that affecting local histone acetylation state would cause some chromatin instability subsequent to transcription that would leave free ssDNA prone to be targeted by AID.
Finally, it should be noted that there was a slight increase in the expression of AID itself in HDAC2–/– cells. Interestingly, ectopic expression of AID has been reported to promote mutation in several transcribed non-Ig genes (Martin et al. 2002; Yoshikawa et al. 2002; Okazaki et al. 2003), and to deregulate targeting specificities of somatic hypermutation in BL2 cells (Woo et al. 2003). Although the increase in AID levels in HDAC2–/– cells was relatively modest (approximately two- to threefold) compared to the overproduction levels in the above-mentioned studies, we cannot exclude that it may have some impact on the targeting of gene conversion. However, some data indicated that enforced expression of AID by retroviral transfection enhanced gene conversion in IgLV with a pattern similar to TSA-treated WT, i.e. with most of the sequences (approximately 70%) harboring sequence changes in CDR1 after 1 month of culture (Seo, Hashimoto, Takeda, et al., unpublished observations). The studies in BL2 cells (Woo et al. 2003) also suggested that the mutation profiles induced by TSA and by AID over-expression share similarities, with an increase of hypermutation at the promoter proximal end of the V region and the constant region. Therefore, it is difficult to conclude that the altered gene conversion pattern in HDAC2–/– is simply due to higher levels of AID.
Overall, our data point to the importance of the function of HDAC2 in the regulation of Ig diversification. Appropriate balance between activities of distinct HDACs (HDAC2 and possibly other TSA-sensitive HDACs that remain to be determined) might significantly contribute to the controlled targeting and promotion of gene conversion. The additive effects observed in TSA-treated HDAC2–/– cultures suggest the existence of distinct HDAC-mediated pathways of regulation. Their understanding may provide new insights into how the cell ensures both maintenance of genomic integrity and target-specific diversification. The ability of DT40 cells for autonomously diversifying Ig genes has also lead to the recent emergence of innovative applications for accelerated selection of monoclonal antibodies (Seo et al. 2005) and protein optimization (Bachl et al. 2007). The finding that the targeting preferences of diversification could be differentially modulated may open the way to the development of new approaches to direct sequence alterations to specific regions of the target gene.
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Experimental procedures |
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The CL18 variant DT40 cells were initially provided by Dr Shunichi Takeda (Buerstedde et al. 1990). The heterozygous HDAC2+/– (clone H2–2.8) and heterozygous HDAC2–/– cells (clones H2–3.1 and H2–3.19) were derived from the parental CL18 as described below. All cultures were performed as described previously (Seo et al. 2006) in IMDM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (JRH), 1% chicken serum (Invitrogen), 50 U/mL penicillin, 50 µg/mL streptomycin (Invitrogen), 55 µM 2-mercaptoethanol (Invitrogen), at 39.5 °C in a 5% CO2 incubator. Media were changed every 1 or 2 days, and the cell density was kept lower than approximately 2 x 106cells/mL. TSA provided by Dr MinoruYoshida (Yoshida et al. 1990) was added in the medium at each passage at the appropriate concentration when indicated.
HDAC2 knockout construct
The BamHI (6949)–SacI (13140) fragment (6.2 kb) of the chicken HDAC2 gene was amplified by PCR from DT40 genomic DNA, digested and cloned into the pBluescriptII SK– vector (Stratagene, La Jolla, CA). PCR primer sequences are provided in Supplementary Fig. S1. Then the StuI (7543)–StuI (10129) fragment (2.6 kb containing part of exon 3 and the entire exons 4 and 5) was deleted and replaced by blunt-ended bsr and puro marker cassettes under control of the β-actin promoter. 30 µg of the resulting plasmid constructs, pWL-HDAC2::bsr and pWL-HDAC2::puro, were linearized by SacI and dissolved in PBS for transfection of approximately 107 DT40 cells by electrophoresis (Bio-Rad Gene Pulser, 550V, 25 µF, Bio-Rad, Hercules, CA). After an overnight culture in non-selective medium at 37 °C, single-clone transformants were selected by limited dilution in 96-well plates at 39.5 °C in selective media (25 µg/mL blasticidin or 0.5 µg/mL puromycin). The heterozygous HDAC2+/– knockout clone H2–2.8 was obtained by transfection of wild-type CL18 with pWL-HDAC2::bsr. Homozygous HDAC2–/– knockout clones H2–3.1and H2–3.19 were obtained by transfection of H2–2.8 with pWL-HDAC2::puro. Targeted integration at the genomic HDAC2 locus was checked by Southern blot after digestion of the genomic DNA by BamHI. Using the PstI-SacI fragment (0.8 kb) of the HDAC2 gene as probe, a 4.1 kb BamHI fragment was revealed as expected when integration had occurred, instead of a 7-kb BamHI fragment revealed when the HDAC2 gene was intact.
Flow cytometry and fluctuation analysis
About 1 x 106 cells were harvested and washed in PBS, 0.3% BSA, stained for surface IgM in 100 µL of 4 µg/mL goat anti-chicken IgM antibody conjugated to FITC (Bethyl, Montgomery, TX), then for DNA with 5 µg/mL propidium iodide to gate out dead cells. The percentage of surface IgM positive cells (sIgM+) was determined using the Epics Elite ESP flow cytometer (Beckman Coulter, Miami, FL).
For fluctuation analysis, single cells were isolated by limited dilution in 96-well plates. Colonies were picked up after 6 days of incubation at 39.5 °C, transferred into 24-well plates and expanded for additional 2 days ("1-week" samples). The samples were then divided and further cultured for one more week in media supplemented with TSA at the indicated concentrations ("2-week" samples). sIgM+ cell population were analyzed by flow cytometry as above.
CFSE dye dilution assay
A total of 2 x 106 cells were harvested, washed, and stained with 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) in 1 mL PBS, 0.1% BSA at 37 °C for 10 min (Molecular Probes, Eugene, OR) following the protocol of the manufacturer. After three washes, the cells were suspended in 10 mL fresh medium and further cultured in usual conditions. For every 24 h, 2 x 106 cells were harvested and stained for surface IgM with R-PE conjugated mouse anti-chicken IgM antibody (SouthernBiotech, Birmingham, AL) and for dead cells with propidium iodide. The fluorescence intensity of gated sIgM+ and sIgM– cells was analyzed at each time-point by keeping constant detection parameters with a Becton–Dickinson FACSort flow cytometer.
Total RNA extraction
Approximately 1 to 2 x 106 cells were centrifuged at 1000 g for 5 min, frozen in liquid nitrogen and stored at –80 °C. Total cellular RNA was isolated with 1 mL TRIzol reagent (Invitrogen) followed by isopropanol precipitation as recommended by the manufacturer. For quantitative expression analysis, RNA integrity and concentration were evaluated using the Agilent 2100 Bioanalyser. Around 1–3 mg of RNA were usually obtained per sample and the concentrations were adjusted to about 20 ng/µL for further experiments.
RT-PCR expression analysis
To test for HDAC2 expression, RT-PCR was performed using the ‘Superscript III One-Step RT-PCR with Platinum Taq Polymerase’ (Invitrogen), in a 25 µL reaction mix including approximately 50 ng of template total RNA and 5 pmol of primers corresponding to nearly a 1-kb fragment of HDAC2 mRNA and of β-actin mRNA (positive control). Primer sequences are in Supplementary Fig. S1. RT-PCR temperature program was set as follows: 30 min of incubation at 55 °C, 2 min denaturation at 94 °C, 25 cycles with 15 s at 94 °C, 30 s at 60 °C, 1 min at 68 °C, then 5 min at 68 °C of final extension.
Real-time quantitative RT-PCR
Nearly 400 ng of total RNA were reverse transcribed using the PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) as recommended by the manufacturer. Real-time RT-PCR was performed with 0.5 µL of the obtained cDNA mix (equivalent to nearly 3 ng starting RNA) using the SYBR Premix ExTaq (Takara) in an ABI PRISM 7300 Fast Real-Time PCR System (Applied Biosystems. Foster City, CA). Primer sequences are provided in Supplementary Fig. S1. Gene expression levels were normalized to β-actin levels.
Genomic DNA extraction
A total of 1 x 105 cells were centrifuged at 1000 g for 5 min and stored as frozen pellets at –80 °C. The samples were thawed and treated using the Mag Extractor MFX-2000 (TOYOBO, Osaka, Japan) with the corresponding Genome Purification Kit according to the protocol of the manufacturer. DNA were recovered in 100 µL sterile H2O.
Ig sequence analysis
The IgL and IgHV region sequences were analyzed as previously described (Seo et al. 2005), with some modifications. One microlitre of genomic DNA (equivalent to 5000 cells) was amplified by PCR using Expand High Fidelity Plus System (Roche, Basel, Switzerland) with the following program: 2 min denaturation at 94 °C, 27 cycles with 30 s at 94 °C, 30 s at 57 °C, 1 min at 72 °C, then 5 min at 72 °C of final extension. The primers used to amplify the rearranged IgLV and IgHV regions are provided in Supplementary Fig. S1. After purification with Qiaquick Gel Extraction Kit (Qiagen, Valencia, CA), the PCR products were cloned into the pCR2.1 TOPO vector (Invitrogen) and sequenced using the M13 universal forward or reverse primers with an ABI 3730xl sequencer (Applied Biosystems). Sequences were aligned to published V pseudogene sequences (Reynaud et al. 1987, 1989) using the BioEdit sequence alignment editor. By visual comparison against the initial sequence before diversification (determined at an early time-point of the culture), we characterized alteration events as follows: "gene conversion" (tract of at least 2 bp changes with perfect sequence identity to a same donor pseudogene), "ambiguous mutation" (single nucleotide change and its flanking nucleotides within a string of at least 5 bp with perfect identity to a pseudogene), "point mutation" (non-templated single nucleotide change with no obvious identity to any known pseudogene), "deletion" or "insertion" (including duplications and apparent template slippage during gene conversion). No distinction was made between ambiguous and non-templated mutations in the IgHV region, since not all pseudogene sequences are known for the heavy-chain.
Chromatin immunoprecipitation assay (ChIP)
ChIP was performed as previously described (Seo et al. 2005) with anti-acetylated histone H4 antibody (Upstate #06-866). Enrichment at the IgLV locus of the active or silent allele was analyzed by real-time PCR using the SYBR Premix ExTaq (Takara) in a ABI PRISM 7300 Fast Real-Time PCR System (Applied Biosystems). Primers were chosen in regions of low diversification frequency (assuming that at the time when samples were taken, not enough sequence diversification has occurred to prevent their annealing) to amplify about 100–200 bp fragments overlapping the IgLV region (see Supplementary Fig. S1). Enrichment levels were normalized to the amplification level in the input chromatin corresponding to one-fifth of the analyzed whole cell extracts.
Western blot analysis
SDS-polyacrylamide gel electrophoresis has been performed by running 15 µL of whole cell extracts (equivalent to nearly 2 x 105 cells) on a Novex 4%–20% Tris–Glycine gel (Invitrogen) using a standard protocol as recommended by the manufacturer. The samples were transferred on an Immobulon PVDF membrane (Millipore, Billerica, MA) and then incubated with 1 : 5000 mouse monoclonal anti-HDAC2 primary antibody (Sigma, St Louis, MO) then 1 : 10 000 HFP conjugated anti-mouse IgG secondary antibody. Presence of HDAC2 protein was revealed using the ECL western blotting detection kit (Amersham Biosciences, Uppsala, Sweden).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kohta{at}bio.c.u-tokyo.ac.jp
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References |
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Received: 1 October 2007
Accepted: 25 November 2007
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CDR1" (black), sequence alterations upstream or overlapping CDR1; "both" (dark gray), alterations in both CDR1 and regions downstream of CDR1; "> CDR1" (light gray), alterations in regions downstream of CDR1 only; "none" (white), no alteration compared to the initial sequence before culture (see text for details). At least two independent subclones were used for each type of culture and the total number of analyzed sequences is indicated above each chart. The results for the TSA-treated WT are representative of experiments performed with either 5 nM or 10 nM of TSA, as no significant difference was observed at these concentrations (see Supplementary Fig. S2 (E–I) for detailed patterns).