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1 Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of Medicine, and 2 Department of Life Science, Frontier Science Research Center, University of Miyazaki, 5200, Kihara, Kiyotake, Miyazaki 889-1692, Japan
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Introduction |
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In eukaryotes, alterations in the chromatin structure not only are involved in regulations of gene expressions, DNA replication, repair, and recombination and so on (Hassig & Schreiber 1997; Wu & Grunstein 2000; Forsberg & Bresnick 2001; Lehrmann et al. 2002; de Ruijter et al. 2003; Yang & Seto 2003; Wang et al. 2004; Huo & Zhang 2005; Mai et al. 2005), but also play a significant role in regulations of early stages of lymphocyte development and differentiation (Agarwal & Rao 1998; Schlissel 2000). The acetylation state of nucleosomal core histones probably induces an open chromatin configuration. The acetylation and deacetylation of core histones should be precisely controlled through cooperative actions of histone acetyltransferase(s) (HATs) and deacetylase(s) (HDACs) (Brownell et al. 1996; Mizzen et al. 1996; Ogryzko et al. 1996; Tauton et al. 1996; Yang et al. 1996).
HATs are grouped into two types (A and B) based on their intracellular distribution and substrate specificity (Nakayama & Takami 2001; Kikuchi et al. 2006). The A type HATs (GCN5, PCAF, MORF, MOZ, TIP60 and so on) are nuclear enzymes generally acting as transcriptional regulators, and the B type HATs (HAT1 and HAT2) are enzymes acetylating newly synthesized histones in cytoplasm. The HAT family members have been reported to be linked to transcription regulations of various cell function-related genes. Recently, using gene targeting techniques for the chicken DT40 B cell line, we established that GCN5 acts as a supervisor in the normal cell cycle progression exhibiting comprehensive control over-expressions of several cell cycle and apoptosis-related genes, probably through alterations in the chromatin configuration (Kikuchi et al. 2005). Moreover, HAT1 is dispensable for replication-coupled chromatin assembly but contributes to recover DNA damages created upon replication blockage (Barman et al. 2006).
HDACs are grouped into three classes (Nakayama & Takami 2001; Kikuchi et al. 2006). HDAC1, 2, 3 and 8 are known as class I HDACs, based on their structural resemblance, nuclear localization and expressions in most cells. Class II HDACs (HDAC4, 5, 6, 7 and 9) are homologues of yeast HDAC HDA1, show cell type-specific expression patterns and are ubiquitously present in both nucleus and cytoplasm. Class III HDACs (SIRT1-7) are homologues of yeast silent information regulator (Sir) 2 and utilize nicotinamide adenine dinucleotide (NAD) as a cofactor for their activity. With regard to HDACs, interesting results have been reported in rapid succession (Hassig & Schreiber 1997; Wu & Grunstein 2000; Forsberg & Bresnick 2001; Lehrmann et al. 2002; de Ruijter et al. 2003; Yang & Seto 2003; Wang et al. 2004; Huo & Zhang 2005). For instance, using trichostatin A (TSA), a specific HDAC inhibitor, it has been reported that histone acetylation regulates transcription of genes controlling terminal B cell differentiation (Lee et al. 2003). To assess individual roles of HDAC members, we have systematically generated a number of homozygous (or conditional) DT40 mutant cell lines, respectively, devoid of specific HDACs (Takami et al. 1999; Takami & Nakayama 2000; Matsushita et al. 2005). Interestingly, HDAC2 controls the amount of IgM H-chain at the steps of transcription of its gene and alternative processing of its pre-mRNA (Takami et al. 1999). In addition, HDAC2 down-regulates IgM L-chain gene promoter activity (Takechi et al. 2002). However, detailed mechanisms of the transcriptional regulations mediated by HDAC2 have been still unclear.
In this study, we showed that the HDAC2-deficiency led to the repression of the HDAC7 gene and activations of the HDAC4, HDAC5 and PCAF genes, which probably caused changes in bulk acetylation levels of several particular Lys residues of core histones. In addition, the HDAC2-deficiency caused repressions of early B cell factor (EBF1), Pax5, Aiolos and Ikaros genes, but activation of the E2A gene. To clarify roles of EBF1, Aiolos and E2A in transcription regulations of IgM H- and L-chain genes, we generated three homozygous DT40 mutant cell lines: EBF1–/–, Aiolos–/– and E2A–/–. Semiquantitative RT-PCR analyzes revealed that EBF1 and Aiolos down-regulate both IgM H- and L-chain genes, whereas E2A up-regulates these two genes. These results, together with others, indicate that HDAC2 controls expressions of IgM H- and L-chain genes, probably through opposite transcriptional regulations of EBF1, Pax5, Aiolos plus Ikaros and E2A genes.
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Results |
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In this study, we usually used three independent HDAC2–/– mutants clones: cl.33-28, cl.33-30 and cl.45-28, all of which exhibited essentially the same phenotypic characteristics. First, we carried out RT-PCR using appropriate primers on total RNA prepared from DT40 and HDAC2–/– cell lines. As shown in Fig. 1A, in the HDAC2–/– mutants, the steady-state mRNA levels of total IgM H-chain and its secreted form were increased, and that of its membrane-bound form was decreased slightly. Moreover, the HDAC2-deficiency merely elevated the expression of IgM L-chain gene. Thus, these results agreed with our previous observations (Takami et al. 1999; Takechi et al. 2002).
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75 kDa) on reduced gel electrophoresis (Fig. 1B, left). On the other hand, most of IgM H-chains from HDAC2–/– mutants, as well as those from DT40 cells, showed the bands with a slower mobility (more than 250 kDa) on native gel electrophoresis (Fig. 1B, right). These results indicated that the majority of artificially accumulated IgM H-chains in the HDAC2–/– mutants exist as a native soluble form to be capable of building a high molecular weight complex with IgM L-chains. In the HDAC2–/– mutants, furthermore, constitutively synthesized IgM H-chains should be accumulated, since not only could the HDAC2 mediated regulatory mechanisms not function any longer, but also the lack of the regulatory mechanisms could be far superior to the capacity to secrete immunoglobulin proteins.
The histone subtype gene families have the inherent ability to compensate for either lack or inactivation of their constituents so as to maintain the greater part of normal levels of their own transcripts, which are definitely based on increased expressions of remaining members (Nakayama & Takami 2001; Kikuchi et al. 2006). To determine whether the HDAC2-deficiency affects gene expressions of remaining HDAC and HAT members inclusive of core histones, RT-PCR was finally carried out. As expected, no changes in the mRNA levels of core histones H2A, H2B, H3 and H4 were observed (Fig. 1A). On the other hand, among HDAC family members examined, the mRNA levels of HDAC4 and HDAC5 were increased (to 150% and 160%), together with the decreased mRNA level of HDAC7 (to 60%), although no changes in those of HDAC1, HDAC3, HDAC8 and HDAC9 were observed (Fig. 1A). Similarly, among HAT family members examined, the mRNA level of PCAF, originally being very low in DT40 cells, was dramatically increased, the increase being close to eightfold, while those of remaining members, GCN5, HAT1, ELP3, MORF (TIP5), MOZ (TIP6), TIP60 and p300, were not changed (Fig. 1A). These results suggested that among the constituents of HDAC and HAT families, HDAC4, HDAC5, HDAC7 and/or PCAF should play, in part, compensatory and/or regulatory roles in chromatin dynamics to maintain a balancing state of acetylation for core histones in the absence or inactivation of HDAC2.
Effects of HDAC2-deficiency on acetylation levels of Lys residues of core histones
The above results concerning increased or decreased expressions of some other HDAC and HAT members due to the HDAC2-deficiency (Fig. 1A) led us to expect that the deficiency may result in considerably altered acetylation patterns of particular Lys (K) residues in the N-terminal tails of core histones. To study this possibility, we performed immunoblotting analysis on HDAC2–/– mutants, using series of available site-specific anti-acetylated core histone antibodies. As shown in Fig. 2A, in the mutants the bulk acetylation levels of core histone H3 at K9, K14, K18, K23 and K27 were increased by 290%, 285%, 185%, 240% and 165%, respectively, whereas the methylation level of K9 remained constant. In the mutants, the bulk acetylation level of core histone H4 only at K5 was increased (to 170%) without alterations at other sites of K8, K12 and K16. Similarly, the acetylated K7 of core histone H2A in the mutants was increased up to 140% with insignificant changes in the acetylation levels of K5 and K9. Finally, in the mutants, the bulk acetylation level of core histone H2B at K16 was decreased, the decrease being to 50%, whereas no changes in the acetylation levels of K5, K12, K15 and K20 were observed.
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Since the HDAC2-deficiency caused changes in two phenotypic properties such as the increased expressions of IgM H- and L-chain genes and altered bulk acetylation levels of various particular Lys residues of core histones H3, H4, H2A and H2B, we expected that the deficiency may alter the chromatin configuration restricted to narrow regions surrounding promoters and/or cis-acting elements of the IgM H-chain gene. Unfortunately, we could not isolate complete genomic DNA clones for chicken IgM H-chain, even after several attempts. However, it was also obvious that the chromatin configuration, including the coding region of the IgM H-chain gene, was somewhat changed in the HDAC2–/– mutants, since the rearranged allele of the gene was more actively transcribed in the mutants than in the control cell line (Fig. 1; Takami et al. 1999). Therefore, we performed a micrococcal nuclease (MNase) sensitivity assay on the nuclei from DT40 and HDAC2–/– cell lines, probing with 32P-labeled RT-PCR amplified fragment, corresponding to a part of IgM H-chain cDNA (from nucleotides 147–458). As shown in Fig. 2B (top), the depletion of HDAC2 had an insignificant effect on the global alterations in chromatin structure. Moreover, when analysed with the labeled IgM H-chain cDNA, MNase digested chromatins from both the cell lines also showed indistinguishable patterns of characteristic nucleosome ladders (Fig. 2B, bottom).
Effects of HDAC2-deficiency on gene expressions of transcription factors
Although a number of transcription factors and coactivators have been reported to be involved in transcription regulation of the IgM H-chain gene, especially in humans and mice (Ernst & Smale 1995), the molecular mechanisms of participations of these factors remained elusive in chickens. Therefore, we tested the possibility that HDAC2 is involved in gene expressions of putative transcription factors that facilitate directly in transcriptional regulation of the IgM H-chain gene. Semiquantitative RT-PCR using appropriate primers was carried out on total RNA prepared from DT40 and HDAC2–/– cell lines (Fig. 2C). Among transcription factors examined, in the mutants the mRNA level of E2A was increased to the extent of 170%. Conversely, the mRNA levels of EBF1, Pax5, Aiolos and Ikaros were decreased dramatically and slightly (to the undetectable level, 60%, 50% and 60%), whereas no changes were detected in the mRNA levels of remaining factors, that is, Oct1, Oct2, OBF1, NF-kB, RelB, YY1, NF-AT, PU.1 and CstF-64. These findings suggested that at least one or some of these altered factors should be involved in transcriptional regulation of the IgM H-chain gene.
Effects of EBF1-deficiency on gene expressions of IgM H- and L-chains
To generate the EBF1-deficient mutant EBF1–/–, we transfected DT40 cells with the pEBF1/bsr vector (Fig. 3A). As expected, after integration of the targeting vector into one EBF1 allele, many stable positive transfectants were selected based on resistance to bsr and signal of the hybridized 8.3 kb KpnI fragment, in addition to the endogenous 6.5 kb KpnI fragment, using the probe EBF1 (Fig. 3B). One of these clones (–/+) was chosen for the second round of transfection with the pEBF1/hisD vector. As expected, in four of several analysed clones (–/–), the probe EBF1 newly hybridized to the 9.1 kb KpnI fragment, in addition to the 8.3 kb KpnI fragment, with disappearance of the endogenous 6.5 kb fragment (Fig. 3B). To confirm whether EBF1 is really disrupted in the corresponding mutants, we measured the steady-state level of EBF1 mRNA by RT-PCR (Fig. 3C). The steady-state level of EBF1 mRNA in the heterozygous mutant (cl.24) was about 50% that as compared to DT40 cells. For the homozygous mutants (cl.24-65, cl.24-83, cl.24-100), no bands were detected.
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180% and 220%). The mRNA level of its membrane-bound form was also increased (to 220%), and this result was not in line with that obtained from the HDAC2–/– mutants (Fig. 1A; Takami et al. 1999), the reason for which is not yet clarified. Moreover, the slightly increased mRNA level of IgM L-chain was observed, the increase being to 110%. These results indicated that EBF1 should down-regulate transcriptions of IgM H- and L-chain genes in the DT40 cell line, and this down-regulation of the IgM H-chain gene agreed in part with the previous result (Akerblad et al. 1996). Effects of Aiolos-deficiency on gene expressions of IgM H- and L-chains
To generate the Aiolos-deficient mutant Aiolos–/–, we transfected DT40 cells with the pAiolos/hisD vector (Fig. 4A). As expected, after integration of the targeting vector into one Aiolos allele, many stable positive transfectants were selected based on resistance to histidinol and signal of the hybridized 7.0 kb KpnI fragment, in addition to the large endogenous KpnI fragment, using probe Aiolos (Fig. 4B). One of these clones (–/+) was chosen for the second round of transfection with the pAiolos/bsr vector. In eight of the analyzed clones (–/–), probe Aiolos newly hybridized to the 6.2 kb KpnI fragment, in addition to the 7.0 kb KpnI fragment, with disappearance of the large endogenous fragment (Fig. 4B). We confirmed the disruption of the Aiolos gene by measuring the steady-state level of Aiolos mRNA by RT-PCR. As shown in Fig. 4C, the steady-state level of Aiolos mRNA in the heterozygous mutant (cl.1) was about 50% that in DT40 cells, and no bands were detected in the homozygous mutants (cl.1-2, cl.1-14, cl.1-25).
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130%, 310% and 110%, respectively. Moreover, the mRNA level of IgM L-chain was also increased slightly to 110%. These results indicated that Aiolos exhibits a negative effect on expressions of IgM H- and L-chain genes. Effects of E2A-deficiency on gene expressions of IgM H- and L-chains
To generate the E2A-deficient mutant E2A–/–, the pE2A/hisD and pE2A/neo vectors were sequentially introduced into DT40 cells (Fig. 5A). As expected, in the clones selected with hisD (–/+), probe E2A newly hybridized to the 7.0 kb HindIII fragment, in addition to the endogenous 6.6 kb HindIII fragment (Fig. 5B). In the case of clones selected with histidinol and neo (–/–) after the second transfection, probe E2A newly hybridized to the 9.7 kb HindIII fragment, with disappearance of the endogenous 6.6 kb HindIII fragment (Fig. 5B). Similar results were obtained with many other clones. To confirm whether E2A is disrupted in the corresponding mutants, we measured the steady-state level of E2A mRNA by RT-PCR. As shown in Fig. 5C, the mRNA level in the heterozygous mutant (cl.68) was about 50% that in DT40 cells, but no bands were detected in the homozygous mutants (cl.57, cl.60, cl.113).
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60% and 30%) in E2A–/– mutant cells. The decreased mRNA level of its membrane-bound form was also observed (to 50%). These results did not agree with those obtained from the HDAC2–/– mutants (Fig. 1A; Takami et al. 1999), the reason for which still remains to be resolved. Moreover, the mRNA level of IgM L-chain was also decreased (to 30%). These results indicated that E2A should up-regulate transcriptions of IgM H- and L-chain genes in the DT40 cell line, and such up-regulations are in line with the results reported in the Abelson pre-B cell clone (Greenbaum et al. 2004), but not with those reported in the DT40 clone 18 cell line (Conlon & Meyer 2006) and the surface Ig+ DT40 AIDR
V– mutant clone (Schoetz et al. 2006). Insignificant effects of deficiencies of other HATs and HDACs on amounts of intracellular IgM H- and L-chains
The HDAC2-deficiency caused not only the increased expressions of HDAC4, HDAC5 and PCAF genes but also the decreased expression of the HDAC7 gene (Fig. 1A), which probably resulted in the alterations in bulk acetylation levels of several Lys residues of core histones (Fig. 2A). To know whether the deficiencies of other HATs and HDACs affect gene expressions of IgM H- and L-chains and/or their amounts, we compared mRNA levels of IgM H- and L-chains and/or total cellular proteins in various homozygous DT40 mutants, devoid of particular members of either HATs or HDACs, by RT-PCR and/or two dimensional (2D)-PAGE followed by the fluorostaining method with SYPRO Red as described (Takami et al. 1999). Detailed information to generate these mutants, except those for HDAC1 and HDAC2 (Takami et al. 1999), HDAC3 (Takami & Nakayama 2000), SIRT1 and SIRT2 (Matsushita et al. 2005), GCN5 and PCAF (Kikuchi et al. 2005) and HAT1 (Barman et al. 2006), will be shown elsewhere. The results obtained from the homozygous HAT and HDAC deficient mutants are briefly summarized in Table 1. The slight deviations on amounts of intracellular IgM H- and L-chains were detected only in the cases of PCAF and GCN5 deficiencies. No changes were detected in remaining single mutants, each devoid of HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT2, HAT1, MOZ, MORF or TIP60 and also in double knockout mutants, respectively, lacking HDAC4/HDAC7 and MOZ/MORF. These findings suggested that among HAT and HDAC members, HDAC2 preferentially participates in transcription regulations of IgM H- and L-chain genes, although we could not yet generate the HDAC5-deficient mutant.
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Discussion |
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Even though both the HDAC2 depletion and, as a result of which, altered amounts of HDAC4, HDAC5, HDAC7 plus PCAF caused the noticeable alterations in the bulk acetylation levels for several particular Lys residues of core histones H3, H4, H2A and H2B (Figs. 1A and 2A), we could not detect alterations in the chromatin configuration, including the coding region of the IgM H-chain gene in the HDAC2–/– mutants, using the IgM H-chain cDNA as a probe (Fig. 2B, bottom). These findings suggest that the chromatin configuration restricted to the region surrounding the IgM H-chain gene in the DT40 cell line is probably kept as an open and active form so as to respond immediately to the appropriate signal(s), because it was derived from the chicken pre-B cell lineage (Buerstedde et al. 1990). However, we cannot exclude the possibility that HDAC2 is directly related to alterations in the chromatin configuration of the IgM H-chain gene, because DT40 cells carry both rearranged and un-rearranged alleles of the gene.
On the other hand, these results also led us to expect that the HDAC2-deficiency may cause the regulation of gene expression of putative transcription factor(s), which should directly control transcription of the IgM H-chain gene. To study this possibility, in the HDAC2–/– mutants, we examined mRNA levels of various transcription factors that had been characterized to be specific for transcription of the IgM H-chain gene and/or to be related to B cell development in other organisms (Ernst & Smale 1995; Liberg et al. 2003; Busslinger 2004; Hagman & Lukin 2005). The HDAC2-deficiency was accompanied with depleted mRNA levels of EBF1, Pax5, Aiolos and Ikaros, and elevated mRNA level of E2A, whereas mRNA levels of all remaining factors examined were maintained constant (Fig. 2C). Thus, HDAC2 plays critical roles in opposite transcriptional regulations, that is, up-regulation of EBF1, Pax5, Aiolos and Ikaros genes and down-regulation of E2A gene.
To explore whether EBF1, Aiolos and E2A really control expressions of IgM H- and L-chain genes, we generated and analyzed three homozygous DT40 mutant cell lines such as EBF1–/–, Aiolos–/– and E2A–/–. The EBF1 depletion could trigger the accelerated expression of the IgM H-chain gene and accumulations of transcripts for total IgM H-chain and its secreted and membrane-bound forms (Fig. 3E). Moreover, the EBF1 depletion caused a little elevation of mRNA level of the IgM L-chain. The Aiolos deficiency also caused mild elevations of mRNA levels of total IgM H-chain and its secreted and membrane-bound forms (Fig. 4E). Similarly, the Aiolos depletion was accompanied with a slight increase of transcript level of the IgM L-chain. Very recently, it has been reported that both Ikaros and Pax5 down-regulate the expression of the IgM H-chain gene (Nera et al. 2006a,b). We have also obtained almost similar results for transcription regulations of IgM H- and L-chain genes in both Pax5 and Ikaros deficient DT40 mutants generated by us (data not shown). To the contrary, interestingly, the E2A deficiency caused certainly decreased mRNA levels of total IgM H-chain and its secreted and membrane-bound forms, as well as that of IgM L-chain (Fig. 5E). These results were coincident with those reported for the E2A-deficient Abelson pre-B cell clones exhibiting considerable reduction in the expression of the IgM H-chain gene (Greenbaum et al. 2004). On the other hand, it has been reported that over-expression of E47 did not cause any significant increase in IgM L-chain mRNA level (Conlon & Meyer 2006). However, this can be interpreted as E47 over-expression causing only a slight increase in the mRNA level of IgM L-chain, probably because the clone 18 DT40 cell line utilized already transcribed its IgM L-chain gene at high levels (Buerstedde et al. 1990). In the surface Ig+ DT40 variant AIDRV– clones, E2A did not influence IgM L-chain gene transcription (Schoetz et al. 2006). Such a discrepancy between our results and the previous ones may be due to the difference in the assay systems (over-expression vs. gene disruption) and the progenitor DT40 clones (DT40 cells vs. surface Ig+ DT40 variant AIDRV– clones) used for the disruption of the E2A gene. Since the mRNA level of the membrane-bound form was decreased in the HDAC2–/– mutants (Fig. 1A; Takami et al. 1999), there is possible existence of an additional HDAC2 related pathway that contributes to transcription regulation of IgM H-chain gene and/or alternative processing of its premRNA.
Based on the results obtained from HDAC2–/–, EBF1–/–, Aiolos–/–, E2A–/– and other HAT and HDAC deficient mutants, together with other results (Akerblad et al. 1996; Greenbaum et al. 2004; Conlon & Meyer 2006; Nera et al. 2006a,b; our unpublished data), we propose a model for the possible impact of HDAC2 in transcription regulations of IgM H and L-chain genes (Fig. 6). Among HAT and HDAC family members, HDAC2 participates preferentially in the control of amounts of IgM H- and L-chains through regulatory pathways based on acetylation status of core histones. Namely, HDAC2 up-regulates remarkably the expression of the EBF1 gene and inversely EBF1 down-regulates expressions of IgM H- and L-chain genes. Similarly, HDAC2 up-regulates expressions of Aiolos, Pax5 and Ikaros genes, and Aiolos down-regulates merely expressions of IgM H- and L-chain genes. Moreover, both Ikaros and Pax5 certainly down-regulate expressions of IgM H- and L-chain genes.
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DT40 cells and all mutants were cultured as described (Takami et al. 1995). At indicated times, cells were counted to determine growth rates.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from exponentially growing DT40 and its subclones (Takami et al. 1999). RT-PCR and semiquantitative RT-PCR using appropriate sense and anti-sense primers listed in Supplementary Table S1 or as per the previous reports (Ahmad et al. 2005; Kikuchi et al. 2005) were performed essentially as described (Kikuchi et al. 2005). The chicken ß-actin or glyceraldehydephosphate dehydrogenase (GAPDH) gene was used as the control. RT-PCR products were subjected to 1.5% agarose gel electrophoresis and analysed using a luminescent image analyzer LAS-1000plus (FUJIFILM). Nucleotide sequences of all RT-PCR amplified products were confirmed by the PCR sequencing protocol as described (Kikuchi et al. 2005).
Immunoblotting
Total proteins were extracted from exponentially growing DT40 and its subclones, subjected to reduced and native gel electrophoreses, respectively, and electrotransferred to polyvinyliden difluoride (PVDF) membranes as described (Kikuchi et al. 2005). The protein-transferred PVDF membranes were blocked with 5% skim milk in Tris buffered saline and probed with anti-chicken IgM H-chain or series of site-specific acetylated histone antibodies as primary antibodies overnight at 4 °C. Antibody binding was detected by using anti-rabbit IgG conjugated with horseradish peroxidase as a secondary antibody and SuperSignal West Pico Chemiluminescent Substrate as a substrate. Data analysis was carried out using a luminescent image analyzer LAS-1000plus (Kikuchi et al. 2005).
Analysis of chromatin configuration
To examine chromatin configuration of the IgM H-chain gene, an MNase digestion assay was carried out as described (Sanematsu et al. 2006). DT40 and HDAC2–/– mutant cells were washed with cold PBS and incubated in NB (10 mM Tris–HCl, pH 8.0, 0.1 mM EDTA, 2 mM magnesium acetate, 2 mM CaCl2, 1 mM DTT, protease inhibitor cocktail (Sigma)) in the presence of 0.2% NP-40. The resultant nuclei were washed with NB twice, suspended at 20 A260/mL in NB and digested at 37 °C for 8 min with 0.008–0.25 U/mL of MNase. The reactions were stopped by adding 50 mM Tris–HCl, pH 8.0, 25 mM EDTA and 0.5% SDS and then DNA was purified by incubation with 200 µg/mL of proteinase K for 60 min at 37 °C, followed by phenol–chloroform extraction and ethanol precipitation. DNA was electrophoresed in a 1.2% agarose gel, stained by EtBr and transferred to a Hybond N +membrane. To detect nucleosome ladders, the resultant blot was probed with 32P-labeled IgM H-chain coding region (the RT-PCR amplified fragment from nucleotides 147–458) and visualized using a Mac BAS-1000 as described (Kikuchi et al. 2005).
Generations of EBF1, Aiolos and E2A-deficient DT40 cell lines
Three cassettes, carrying bsr, hisD and neo, respectively, transcribed by the chicken ß-actin promoter were obtained as described (Takami & Nakayama 2000). Partial genomic EBF1, Aiolos and E2A DNA fragments were obtained from chicken genomic DNA by means of PCR based on nucleotide sequences from the WEB BURSAL database and confirmed by the PCR sequencing protocol (Kikuchi et al. 2005; Sanematsu et al. 2006). Using these cassettes and genomic EBF1, Aiolos and E2A fragments obtained, we constructed targeting vectors for disruptions of EBF1, Aiolos and E2A genes as follows.
To obtain the pEBF1/bsr or pEBF1/hisD vector for the disruption of EBF1, we ligated the 5'-arm, a 1.5 kb PCR fragment (obtained using sense primer 5'-ATGTTTGGGATCCAGGAAAGCATCCTGCGG-3' from exon 1 and anti-sense primer 5'-TTCTCCACGAAGCCCACGAAGGCGGTGC-3' from exon 2) to the 5'-end of the cassette carrying bsr or hisD, and then the 3'-arm, a 5.3 kb PCR fragment (obtained using sense primer 5'-TATGAAGGCCAAGACAAGAACCCCGAAATG-3' from exon 4 and anti-sense primer 5'-CACTGGATCTGATGGAGTCTCATTTTGGTT-3' from exon 5) to the 3'-end of either vector. In the resultant targeting vectors, therefore, the genomic sequences corresponding to exons 2–4 of EBF1 were replaced with the bsr or hisD-carrying cassette.
To obtain the pAiolos/hisD or pAiolos/bsr vector for the disruption of Aiolos, we ligated the 5'-arm, a 2.4 kb PCR fragment (obtained using sense primer 5'-CCGTCGACGTTAAAGTGAAAAGTGAATACG-3' from exon 3 and anti-sense primer 5'-AAGGATCCACTCACACTTGTAGGGCTTCTC-3' from exon 5) to the 5'-end of the cassette carrying hisD or bsr, and then the 3'-arm, a 4.0 kb PCR fragment (obtained using sense primer 5'-TTGGCGCGCCAAGGTCGAGATGGGGACTGA-3' from exon 6 and anti-sense primer 5'-CGCTCGAGTACACGAAGGACGAGTTGTAAT-3' from exon 7) to the 3'-end of either vector. In the resultant targeting vectors, therefore, the genomic sequences corresponding to exons 5–6 of Aiolos were replaced with the hisD or bsr-carrying cassette.
To obtain the pE2A/hisD or pE2A/neo vector for the disruption of E2A, we ligated the 5'-arm, a 2.1 kb PCR fragment (obtained using sense primer 5'-AGCTGTCGACACGGGCTCCACAACTCGCCAG-3' from exon 7 and anti-sense primer 5'-GTCAGGATCCACTGGGATGTGCCTGGGAACA-3' from exon 11) to the 5'-end of the cassette carrying hisD or neo, and then the 3'-arm, a 4.8 kb PCR fragment (obtained using sense primer 5'-AGCTGGCGCGCCAGGCAGCAGGACACGTACAGT-3' from exon 13 and anti-sense primer 5'-GTCACTCGAGAGAAGCTGAAAGCGTCATCTG-3' from exon 16) to the 3'-end of either vector. In the resultant targeting vectors therefore the genomic sequences corresponding to exons 11–13 of E2A were replaced with the hisD or neo-carrying cassette.
Transfection was carried out essentially as described (Buerstedde & Takeda 1991; Takami et al. 1995). To obtain EBF1–/– mutants, transfectants with pEBF1/bsr vector were selected in medium containing 15 µg of bsr/mL. We transfected the pEBF1/hisD vector into clones, in which one of two EBF1 alleles had already been disrupted and selected stable transfectants in medium containing 15 µg of bsr and 0.8 mg of histidinol/mL, respectively. To disrupt Aiolos, the pAiolos/hisD and pAiolos/bsr vectors were used instead of the pEBF1/bsr and pEBF1/hisD vectors. To disrupt E2A, the pE2A/hisD and pE2A/neo vectors were used instead of the pEBF1/bsr and pEBF1/hisD vectors, and then neomycin (G-418) instead of bsr was used at a concentration of 2 mg/mL.
Southern blotting
Genomic DNA was digested with indicated enzymes, separated in a 0.8% agarose gels, transferred to Hybond N +membranes and then hybridized with 32P-labeled probe EBF1, Aiolos or E2A as described (Takami et al. 1999).
Probe EBF1, 5'-end fragment of intron 5 of EBF1, comprised the 0.5 kb fragment derived from a PCR fragment (obtained using sense primer 5'-TGTTGTGACAAGAAAAGCTGTGGCAACCAA-3' and anti-sense primer 5'-GGATTGCATCCAACAGAACTACTGTAGC-3' from the intron). Probe Aiolos, 3'-end fragment of exon 7 of Aiolos, comprised the 0.5 kb fragment derived from a PCR fragment (obtained using sense primer 5'-AAGCCATCAACAACGCCATCTCCTTCCT-3' and anti-sense primer 5'-CTATTTGAGCAGCACACGGTGC-3' from the exon). Probe E2A, 3'-end fragment of intron 6 of E2A, comprised the 0.5 kb fragment derived from a PCR fragment (obtained using sense primer 5'-GTCAGAATTCGACTACAGCAGGGATCCGGCT-3' and anti-sense primer 5'-GTCAGGATCCTGAATGCAGCTGGTAGTTCTG-3' from the intron).
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Acknowledgements |
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* Correspondence: E-mail: tnakayam{at}med.miyazaki-u.ac.jp
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Received: 25 September 2006
Accepted: 10 December 2006
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