Decreased E12 and/or E47 Transcription Factor Activity in the Bone Marrow As Well As in the Spleen of Aged Mice

Mice

Male and female young (2–4 mo of age) and old (24–27 mo of age) BALB/c mice were obtained from the National Institute on Aging (Bethesda, MD).

Splenic B cell purification

B cells were isolated from the spleens of young and old mice. Briefly, cells were washed twice with medium (RPMI 1640; Life Technologies, Gaithersburg, MD) and incubated (10⁸ cells/ml) for 20 min at 4°C with 100 μl of anti-B220 Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the MiniMacs protocol (Miltenyi). Cells were then purified using magnetic columns. At the end of the purification procedure, cells were found to be almost exclusively (85–90%) B220 positive by cytofluorimetric analysis. After the isolation procedure was ended, cells were maintained in serum-free medium for 3 h at 4°C to minimize potential effects of anti-B220 Abs on B cell activation.

B cell culture

B cells were cultured in complete medium (RPMI 1640, supplemented with 10% FCS, 10 μg/ml penicillin-streptomycin and 2 mM l-glutamine). For the determination of the optimal culture conditions, cells (2 × 10⁴ or 2 × 10⁵ in 200 μl of complete medium) were stimulated for 24, 48, or 72 h in 96-well culture plates with LPS (0.1, 1 or 10 μg/ml; Sigma-Aldrich, St. Louis, MO). In the last 4 h of incubation, cultures were pulsed with 1 μCi of [³H]thymidine to determine cell proliferation. Our previous results (34) indicate that optimal B cell proliferation is induced when 1 × 10⁶ cells/ml in a final volume of 200 μl are activated by 10 μg/ml LPS for 48 h. Doses of 0.5 and 2 × 10⁶ cells/ml in a final volume of 200 μl were also evaluated but yielded lower B cell proliferation than did the dose of 1 × 10⁶ cells/ml. For the preparation of nuclear and cytoplasmic extracts, B cells (1 × 10⁶/ml) were stimulated for 48 h in six-well culture plates with 10 μg/ml LPS. These conditions were optimal for cell proliferation. After this time, cells were lysed, and extracts were prepared.

Bone marrow cell culture and Ab staining

Bone marrow cells (3 × 10⁵) from 28 pairs of young and old mice were analyzed for surface CD43, B220, and IgM. Briefly, cells were incubated with PE-labeled anti-CD43 Abs (1/40 diluted; BD PharMingen, San Diego, CA), CY-conjugated anti-B220-Abs (1/40 diluted; BD PharMingen), and FITC-labeled anti-μ Abs (1/100 diluted; Jackson ImmunoResearch Laboratories, West Grove, PA) in FACS buffer (HBSS, buffered in 0.02% sodium azide and 0.1% BSA) for 30 min on ice, using the protocol described elsewhere (26). After staining, cells were fixed with 0.25% paraformaldehyde. Samples of 10⁴ cells were analyzed on a LSR flow cytometer (BD PharMingen) using a logarithmic amplification. The percentage of pre-B cells (IgM⁻CD43⁻B220^low) from total nucleated bone marrow cells was significantly decreased by aging in 23 of 28 mice, similar to previous results (26, 33). Thus, old mice were classified as severely depleted mice (>80% loss in pre-B cells) or moderately depleted mice (20–80% loss in pre-B cells). Of 28 mice, 5 were nondepleted. Table I⇓ summarizes the results obtained with these mice. Expanded populations of pro-B/pre-B cells from young and old mice were obtained as previously described (26, 27). Briefly, bone marrow cells were cultured in six-well culture plates at the concentration of 10⁶/ml in complete medium in the presence of 5 ng/ml murine IL-7 (BioSource International, Camarillo, CA). Seven days later, nonadherent cells were harvested, analyzed by flow cytometry, and lysed; nuclear and cytoplasmic extracts were prepared. The expansion of pro-B/early pre-B cells with IL-7 was decreased significantly in the bone marrow from old as compared with young mice beginning at day 3 after culture initiation and continuing through day 7. At day 7, we observed the maximum difference in expansion between young and old mice (26). After day 7, a great proportion of dead cells were found in both young and old mice.

Preparation of nuclear and cytoplasmic extracts and total cell lysates

Nuclear and cytoplasmic extracts were prepared from cultured bone marrow or spleen cells essentially as previously published (35); briefly, cells were harvested and centrifuged in an Eppendorf 5415C microfuge (Brinkmann Instruments, Westbury, NY; New York; 2000 rpm, 5 min). The pellet was resuspended in 30 μl of solution A containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.0 mM EDTA, 1 mM DTT, 1.5 mM MgCl₂, 1 mM PMSF, 1 tablet of protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany), and Nonidet P-40 (0.1%); briefly vortexed; and centrifuged (8000 rpm, 5 min, 4°C). The supernatant containing the cytoplasmic extract was removed, and the pellet containing the nuclei was resuspended in solution B containing 20 mM HEPES (pH 7.9), 0.1 mM EDTA, 1 mM DTT, 1.5 mM MgCl₂, 2 mM PMSF, 1 tablet of protease inhibitor mixture, and glycerol (10%). The lysate was incubated on ice for 20 min, protein sonicated for a few seconds, and centrifuged (14,000 rpm, 15 min, 4°C). Aliquots of the nuclear extract were stored at −80°C. For the preparation of total cell lysates, cultured cells were harvested and centrifuged in an Eppendorf 5415C microfuge (2000 rpm, 5 min). The pellet was resuspended in 30 μl of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 1 tablet of protease inhibitor mixture (Boehringer Mannheim), and 1 mM PMSF; briefly vortexed; and centrifuged (14,000 rpm, 15 min, 4°C). Protein content was determined by the Bradford assay.

Preparation of the DNA probes

The μE5 probe was prepared as follows: 100 μl of each single strand (26 bp), at a concentration of 100 ng/μl, were annealed volume for volume at temperatures of 85°C (2 min), 65°C (15 min), 37°C (15 min), and 25°C (15 min) and on ice (15 min) and then end labeled for 40 min at 37°C, using T4 DNA polynucleotide kinase in the presence of 1 μl of [γ-³²P]ATP. The probe was then purified over a Sepharose G-25-50 column (Sigma-Aldrich). The ku probe was prepared as follows. A double strand ku DNA fragment (56 bp), 10 pmol of the 56-oligomer, was end labeled for 30 min at 37°C with T4 DNA polynucleotide kinase in the presence of 2 μl of [γ-³²P]ATP, then incubated with 20 pmol of the complementary oligonucleotide at 85°C for 5 min, and subsequently cooled at room temperature. The probe was then purified over a Sepharose G-50-80 spin column (Sigma-Aldrich). The sequences of the probes were: 5′-CCC-GGC-GCGGGG-GCG-ATT-TCG-AGT-CA-3′ (μE5, present in the IgH intronic enhancer) (36) and 5′-GAT-CAG-TGA-TGG-AGT-TGG-CCA-CTC-CCTCTC-TGC-GCG-CTC-GCT-CGC-TCA-CTG-AGG-CC-3′ (ku). The 56-oligomer sequence was constructed by modifying the sequence originally described (37) to obtain optimal ku binding to DNA, as heterodimer ku 70/80 (38).

EMSA

A gel mobility shift assay to determine DNA binding of E47 and of ku was performed as follows. The radiolabeled DNA probe was incubated with 10 μg of nuclear extract in the presence of 0.5 μg of poly(dI-dC) or of the circular plasmid pUC9 as nonspecific competitors for μE5 and ku, respectively. The reaction was performed at room temperature in 15 μl of binding buffer (10 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1 mM DTT, 1 mM PMSF, 6% glycerol). To supershift E47 and/or E12, the nuclear extracts were preincubated with rabbit anti-mouse polyclonal E47 (2 μl; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse polyclonal E12 (2 μl; Santa Cruz Biotechnology) Abs, before the addition of the radiolabeled probes. These Abs were the best supershifting Abs, compared with other anti-E47/anti-E12 Abs (BD PharMingen), based on the evidence that they completely prevented the binding of the μE5 complexes in IL-7-expanded pro-B/pre-B cells, similar to the anti-E47 and anti-E12 mAbs of BD-PharMingen, but could be used at a concentration of 50-fold less protein concentration (4-fold less supplied concentration). To supershift the ku 70/80 heterodimers, the nuclear extracts were preincubated with the polyclonal anti-ku 80 (2 μl; Serotec, Oxford, U.K.), before the addition of the radiolabeled probes. The samples were electrophoresed in 5% polyacrylamide gel at 175 V for 3 h at room temperature. The gels were dried on Whatman 3M paper and exposed to Kodak x-ray films (Eastman, Rochester, NY) at −80°C overnight.

Western blotting

For the evaluation of the amounts of E47 and ku 80 proteins in the cytoplasm and in the nucleus of splenic B cells, cytoplasmic and nuclear extracts at equal protein concentration were denatured by boiling for 4 min in Laemmli sample buffer and subjected to SDS-PAGE using 4–12% polyacrylamide gel under reducing conditions. Proteins were then electrotransferred onto nitrocellulose filters (Bio-Rad, Hercules, CA). Nonspecific sites were blocked by incubation of the membranes with PBS-Tween (1× PBS containing 0.05% Tween 20) containing 5% milk for 1 h at room temperature (blocking solution). Filters were incubated with mouse monoclonal anti-E47 Abs (1/400 diluted, BD PharMingen), mouse monoclonal anti-E12 Abs (1/400 diluted, BD PharMingen), rabbit polyclonal anti-ku 80 Abs (1/500 diluted; Serotec), goat polyclonal anti-Ikaros Abs (1/200 diluted; Santa Cruz Biotechnology), or hamster monoclonal FS1 anti-λ5 Ab (1/5 diluted; gift from Dr. J. Jongstra). These anti-E47 and anti-E12 Abs gave the best signal by Western analyses. Mouse monoclonal anti-β-actin (1/8000 diluted; Sigma-Aldrich) and mouse monoclonal anti-histone H-1 (1/200 diluted; Santa Cruz Biotechnology) were used to detect β-actin and histone H-1, respectively, as loading controls. After overnight incubation with the primary Ab, immunoblots were incubated with the appropriate HRP-labeled secondary Abs (Rockland, Gilbertsville, PA; 1/16,000 diluted, for mouse primary Abs; Santa Cruz Biotechnologies, 1/200 diluted, for rabbit primary Abs; Santa Cruz Biotechnology 1/200 diluted, for goat primary Abs) for 3 h at 4°C, developed by enzyme chemiluminescence, and exposed to CL-XPosure Film (Pierce, Rockford, IL).

DNA binding of E12 and/or E47 are decreased in both bone marrow and spleen of aged mice

Nuclear extracts of whole bone marrow cells from young and old mice, as well as of pro-B/pre-B cells from young and old mice, in vitro expanded with IL-7 for 7 days, were prepared and analyzed by EMSA, using the μE5 sequence as probe. Results were obtained at day 7 because it is very difficult to measure the levels of E2A in IL-7 cultures, at least before day 6, because the small number of cells recovered is not sufficient to allow Western analyses. In addition, the ratios of pro-B vs pre-B cells change during culture, pro-B and pre-B cells express different levels of E2A (12), and therefore cultured cells would have to be sorted before analysis. Results in Fig. 1⇓A show that the sensitivity of our EMSA analysis can measure DNA binding only in nuclear extracts from IL-7-stimulated pro-B/pre-B cells, but not from whole bone marrow cells. Typically, IL-7-stimulated bone marrow results in about a 10× expansion of precursor cells, which gives pro-B cells (∼80%) and early pre-B cells (∼20%) (26). We have previously shown a reduction in pro-B/pre-B cells of old bone marrow cells expanded on IL-7 culture as compared with young bone marrow (26, 33) and a decreased protein level of E12/E47 in old IL-7 blasts (26). Here we show that IL-7-stimulated pro-B/pre-B cells from old mice exhibit a significantly reduced ability to bind the μE5 sequence compared with young mice (Fig. 1⇓A). Both E12 and E47 are in the DNA-binding complex in these expanded pro-B/pre-B cells, as indicated by the use of anti-E12 and anti-E47 Abs in the supershift in Fig. 1⇓A.

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FIGURE 1.

E47/E12 DNA binding is decreased in old bone marrow (BM) precursor B cells and splenic B cells. A, IL-7 blasts from young (Y) but not from old (O) bone marrow show high levels of E12/E47 DNA binding. Untreated young bone marrow cells also do not show activity. Nuclear extracts from the same numbers (10⁷) of whole bone marrow cells or IL-7-expanded pro-B/pre-B cells were run in EMSA (10 μg/lane). EMSA with whole bone marrow cells has been repeated twice, whereas EMSA with IL-7 blasts has been repeated eight times, in all cases giving comparable results. NS, nonspecific bands. Anti-(α)-E47, anti-E12, supershift, described in Materials and Methods. B, E47 DNA binding is up-regulated in splenic B cells from young and, to a significantly lesser extent, old mice. Splenic B cells (10⁶ cells/ml) from one young and one old mouse were left unstimulated or were stimulated with 10 μg/ml LPS for 24, 48, and 72 h. Nuclear extracts from the same numbers of cultured B cells were prepared and run in EMSA (10 μg/lane). E47/E47 complexes can form single (B) or multiple (A) protein-DNA complexes, the number of which depends on the concentration of the transcription factor in the nuclear extract. Films were scanned and analyzed using Scion Image for Windows (Scion, Frederick, MD). Integrated areas under the densitometric curves for each band were used as estimates of DNA binding. Vertical columns represent the fold increase ± SE in E47 DNA binding as compared with those of young, unstimulated controls, taken as 1. Results are the means of the densitometric estimates ± SE from four young and four old mice independently evaluated. Young values were: 1 (unstimulated); 5.3 ± 2.1 (24 h); 8.1 ± 2.4 (48 h); and 4.3 ± 2.8 (72 h). Old values were: 0.6 ± 0.1 (unstimulated); 2.3 ± 0.9 (24 h); 3.43 ± 1.6 (48 h); and 1.8 ± 0.7 (72 h). At 48 h of stimulation, the activation is maximal for young and old cells, and the difference between young and old mice is significant at p < 0.05, as determined by the two-tailed Student t test. □, Young; ▪, old. C, E2A DNA binding in IL-7 blasts and LPS-activated splenic B cells is equivalently reduced in the same aged individual mice. Nuclear extracts from equal numbers (10⁷) of IL-7-expanded pro-B/pre-B cells or splenic B cells stimulated with LPS for 48 h were run in EMSA (10 μg/lane). Films were scanned and analyzed as described in B. In IL-7 blasts, the levels of DNA binding of old vs young were 1–13% in seven of eight mice, with the exception that in one pair of mice (pair 4), old DNA binding was 83% of young controls. This mouse was the only one not to be depleted in pre-B cells (with 11.5% pre-B cells) as evaluated by flow cytometry. In LPS-activated splenic B cells, the levels of old DNA binding were 2–40% compared with young controls in seven of eight mice; and similar to IL-7 blasts, in pair 4, DNA binding was 81% of young controls. D, DNA binding of ku 70/80 is not modified by B cell activation or by aging. The same nuclear extracts shown in B were run in EMSA (10 μg/lane) using a 56-oligonucleotide probe that binds the DNA repair enzyme ku 70/80. The multiple bands are created by one, two, or more multimers of ku binding to DNA. S, Supershift performed with the anti-ku 80 Ab (see Materials and Methods).

To determine the kinetics of E47 DNA-binding in mitogen-activated splenic B cells from young or old mice, nuclear extracts from splenic B cells (1 × 10⁶/ml), activated with 10 μg/ml LPS for 24, 48, or 72 h, were prepared and analyzed by EMSA, using the μE5 probe. Results in Fig. 1⇑B show measurable, although low, E47 DNA binding in unstimulated splenic B cells from young mice. Activation of B cells from young mice with LPS up-regulates E47 DNA binding at all times of stimulation, with the highest level of E47 activation attained after 48 h in the presence of LPS. In old mice, the endogenous E47 DNA binding is lower than that in unstimulated young spleen cells in 13 of 20 (65%) mice individually tested. Additionally, activation of B cells from old mice with LPS induces a lower increase in E47 DNA binding, as compared with that seen in young mice, the fold increase (relative to unstimulated young) in young vs old being 5 vs 2 at 24 h, 8 vs 3 at 48 h, and 4 vs 2 at 72 h. Thus, in old mice also, the highest level of E47 activation was reached after 48 h in the presence of LPS. At 0 time points (endogenous), all of four old mice had lower amounts of E47 DNA binding. The stimulated values (e.g., at 48 h) were lower (also when the stimulation index was calculated compared with the age-matched 0 time point control) for two of four old mice. Therefore, the fold increase of old stimulated mice, relative to unstimulated old, is 3.8 at 24 h, 5.7 at 48 h, and 3.0 at 72 h. Moreover, in other cultures set up for 48 h only, in 7 of 20 old mice (35%) in which the endogenous E47 DNA binding was comparable with that of young mice, the LPS-induced E47 DNA binding was also increased over that of the unstimulated controls, but still lower than that in young mice. These results of lower endogenous E47 DNA binding in aged splenic B cells (2- to 4-fold) have been reproduced in many other experiments. Additionally, the fold stimulation seen at peak times of stimulation (i.e., 48 h with anti-CD40 and IL-4) has also been shown to be always lower in aged vs young splenic B cells (2- to 4-fold) (Ref. 34 and our unpublished results).

Results in Fig. 1⇑C, showing 8 of 20 pairs of mice in this study, indicate a comparable age-related reduction in the ability of nuclear extracts from both IL-7 blasts and LPS-stimulated splenic B cells from the same old mice to bind the μE5 sequence. Even if the age of the mice is toward the end of BALB/c life expectancy (up to 30 mo), we still see in that range of ages old mice that are not depleted in pre-B cells (Fig. 1⇑C, pair 4, where the old mouse is 25 mo old). We do not know when the reduction of E2A-encoded factors is first observed. However, we were able to see decrease in E2A-binding activity in 18-mo-old mice (data not included). From the data published elsewhere (26), altered expression of E2A in old mice is completely correlated with reduction of surrogate light chains observed in in vitro expanded pro-B/early pre-B cells. This in turn contributes to the decreased B lymphopoiesis; therefore, we can expect that the decline in E2A expression likely precedes that of pre-B cell depletion. When the same nuclear extracts were analyzed by EMSA with a 56-oligonucleotide probe, previously described to bind the DNA repair enzyme ku 70/80, no effects of aging or even of LPS stimulation were observed (Fig. 1⇑D). This is consistent with previous reports that ku 70/80 is unaffected during aging in human PBMC (38).

The DNA-binding complex is different in B cell precursors and in mature splenic B cells

We then examined the presence of E47 and/or E12 in the DNA-binding complexes in splenic B cells from young and old mice. Results in Fig. 2⇓ show that in both young and old mice, anti-E47 Abs effectively prevent DNA binding from splenic nuclear extracts, whereas anti-E12 Abs do not modify the DNA binding in the complex, confirming previous reports that only E47/E47 complexes bind DNA in mature B cells (2). Interestingly, and in contrast to splenic B cells, either anti-E47 or anti-E12 Abs were able to completely supershift the band due to DNA binding of E12/E47 proteins in IL-7-blasts, suggesting that the composition of E complexes able to bind DNA differs in B cell precursors (Fig. 1⇑A) as compared with mature B cells. Specificity was shown by the ku probe not being shifted/removed by anti-E47 Abs and by anti-class I bHLH transcription factor Abs not preventing the binding to the μE5 probe (data not shown).

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FIGURE 2.

Only E47/E47 complexes bind DNA in splenic B cells. Splenic B cells (10⁶ cells/ml) were left unstimulated or they were stimulated with 10 μg/ml LPS for 48 h. Nuclear extracts from same numbers of unstimulated and LPS-activated B cells were prepared and run in EMSA (10 μg/lane), in the presence or absence of anti-E47 and anti-E12 Abs. This experiment has been repeated five times, giving comparable results.

E47 expression is up-regulated by B cell activation

The expression of the E2A-encoded proteins by Western blot in total cell lysates from IL-7-expanded pro-B/pre-B cells shows that the levels of E47 and E12 proteins are decreased in old pro-B/pre-B extracts (Fig. 3⇓A). Western analysis was also performed for Ikaros and mouse λ5 proteins. Results show that Ikaros is present at comparable levels in IL-7-expanded pro-B/pre-B cells from young and old mice. Mouse λ5, as already demonstrated previously (26), is decreased by aging, although in this sample to a lesser degree than normally seen in aged pro-B/pre-B cells.

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FIGURE 3.

E47/E12, but not other proteins, are down-regulated in IL-7-expanded pro-B/pre-B cells and LPS-activated splenic B cells by Western blot analysis. A, Total lysates from IL-7-expanded bone marrow pro-B/pre-B cells (10⁶ cells/ml) from young and old mice were prepared and run in Western blot. The titration is 2, 4, and 8 μg of cell lysate/lane. E47, E12, Ikaros, λ5, and β-actin were detected with specific Abs. B, LPS-activated splenic B cells from old mice show reduced E47 expression, but normal ku 80 expression, compared with young mice. Splenic B cells (10⁶ cells/ml) were left unstimulated or they were stimulated with 10 μg/ml LPS for 24, 48, and 72 h. Nuclear extracts from equal numbers of unstimulated and LPS-activated B cells were prepared and run in Western blot (8 μg/lane). E47, ku 80, and histone H-1 were detected with specific Abs. Films were scanned and analyzed as described in Fig. 1⇑B. Vertical columns represent the fold increase ± SE in E47 DNA binding compared with those of young, unstimulated controls, taken as 1. Results are the means of the densitometric estimates ± SE from two young and two old mice independently evaluated. Young values were: 1 (unstimulated); 1.5 ± 0.2 (24 h); 4.3 ± 0.3 (48 h); and 0.6 ± 0.3 (72 h). Old values were: 0.5 ± 0.04 (unstimulated); 1.4 ± 1.0 (24 h); 1.7 ± 0.3 (48 h); and 1.3 ± 1.0 (72 h). At 48 h of stimulation, the difference between young and old mice was significant at p < 0.05, as determined by the two-tailed Student t test. □, Young; ▪, old.

Because DNA binding of E47 is up-regulated in splenic B cells from young and, to a lesser extent, from old mice by LPS, we examined the levels of E47 protein by Western blotting in nuclear extracts of activated splenic B cells from young and old mice. Results in Fig. 3⇑B show that E47 protein is present in the nuclear extracts of unstimulated splenic B cells from young mice. The amount of E47 increases after 24 and 48 h and decreases after 72 h of stimulation in the presence of LPS. In the nuclear extracts of unstimulated B cells from old mice, E47 is present to a lesser extent than in young mice, it increases after 24 and 48 h of stimulation, and it decreases slightly at 72 h. The fold increase in young vs old after LPS stimulation is 1.45 vs 1.44 at 24 h, 4.33 vs 1.69 at 48 h, and 0.6 vs 1.03 at 72 h. The levels of E47 expression were significantly higher in young than in old mice at all stimulation times. These results indicate that E47 DNA binding correlates with E47 protein expression (compare Fig. 3⇑B with Fig. 1⇑B). In Fig. 3⇑B, histone H-1 was used as loading control for the nuclear extracts, because histone H-1 is almost exclusively present in nuclear as opposed to cytoplasmic extracts in splenic B cells and is not modified by aging or stimulation (34). Again, ku 80 is also unaffected by aging and/or by LPS stimulation.

E12 is expressed at lower levels, as compared to E47, in LPS-stimulated splenic B cells from both young and old mice and is down-regulated by aging

To examine whether E12 is modulated by aging in splenic B cells, nuclear extracts from splenic B cells, LPS activated during a period of 48 h, were analyzed for E47 and E12 protein expression by Western analysis (Fig. 4⇓). Results show that, unlike E47, E12 appears to be present at very low levels in LPS-activated splenic B cells from young mice. Aging significantly impairs the expression of both proteins in nuclear extracts of LPS-activated splenic B cells.

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FIGURE 4.

Splenic B cells express predominantly E47 compared with E12. Splenic B cells (10⁶ cells/ml) were stimulated with 10 μg/ml LPS for 48 h. Nuclear extracts from LPS-activated B cells from young and old mice were prepared and run in Western blot. The titration were 2, 4, and 8 μg of cell lysate/lane. E47, E12, and histone H-1 were detected with specific Abs. Films were scanned and analyzed as described in Fig. 1⇑B. Comparing the two lanes loaded with 8 μg/lane each, E12 was found to be 7- to 10-fold less expressed than E47 in LPS-activated B cells from young mice in three independent experiments.

Only nuclear extracts of splenic B cells, whereas both nuclear and cytoplasmic extracts of IL-7-expanded pro-B/pre-B cells exhibit DNA binding

We next addressed whether the different DNA binding of E47 seen in young and old mice may occur through different cellular compartmentalization of this E2A-encoded protein. To this purpose, nuclear and cytoplasmic extracts of LPS-activated splenic B cells as well as IL-7 blasts from both young and old mice were analyzed in EMSA and Western experiments. Results in Fig. 5⇓A indicate that only the nuclear extracts of unstimulated and LPS-activated splenic B cells from young mice exhibit DNA binding; this result correlates with Western data showing that nuclear extracts of LPS-activated splenic B cells exhibit higher expression of E47 than do cytoplasmic extracts (Fig. 5⇓B). In this experiment, cytoplasmic and nuclear extracts of B cells from young and old mice show comparable levels of β-actin. Although β-actin must be present at higher concentrations in the cytoplasm than in the nucleus, β-actin is found also in the nucleus, and the possible contamination of nuclear extracts is irrelevant to our conclusions, because cytoplasmic extracts of splenic B cells do not display DNA binding. B cells from old mice exhibited neither DNA-binding nor E47 expression in these experiments, but in other studies (see Fig. 1⇑C), some (four of eight) nuclear extracts from old mice were also able to show DNA binding. Conversely, in pro-B/pre-B cells, at least from young mice, both nuclear and cytoplasmic extracts display DNA binding and comparable levels of E47 (Fig. 6⇓). Because in almost all IL-7 nuclear and cytoplasmic extracts neither DNA binding nor expression by Western analysis could be seen for E47/E12, we were not able to determine whether both nuclear and cytoplasmic extracts had E47/E12.

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FIGURE 5.

LPS-stimulated splenic B cells show E47 expression and DNA binding only in the nucleus. A, Only nuclear extracts of LPS-stimulated splenic B cells from young mice exhibit DNA binding. In this particular experiment, old mice do not exhibit DNA binding either in nuclear (nu) or cytoplasmic (cy) extracts. Cytoplasmic and nuclear extracts of splenic B cells, unstimulated or stimulated with LPS for 48 h, were run in EMSA (10 μg/lane) in the presence (supershift) or absence of anti-E47 Abs. This experiment has been repeated three times, giving comparable results. NS, Nonspecific bands. B, In LPS-stimulated splenic B cells, E47 is expressed almost exclusively in the nucleus. Splenic B cells (10⁶ cells/ml) were stimulated with 10 μg/ml LPS for 48 h. Cytoplasmic and nuclear extracts from LPS-activated B cells from young and old mice were prepared and run in Western blot. The titration was 2, 4, and 8 μg of cell lysate per lane. E47, β-actin, and histone H-1 were detected with specific Abs. Films were scanned and analyzed as described in Fig. 1⇑B. Comparing the two lanes loaded with 8 μg/lane each, E47 was found to be 10-fold less expressed in the cytoplasm than in the nucleus of LPS-activated B cells from young mice.

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FIGURE 6.

IL-7-expanded pro-B/pre-B cells show E47 and E12 expression and DNA binding in both the nucleus (nu) and cytoplasm (cy). A, Both nuclear and cytoplasmic extracts of IL-7-expanded pro-B/pre-B cells exhibit DNA binding. Cytoplasmic and nuclear extracts of IL-7-expanded pro-B/pre-B cells were run in EMSA (10 μg/lane) in the presence or absence of anti-E47 and anti-E12 Abs. This experiment has been repeated six times, giving comparable results. Anti-E47, anti-E12, supershift, described in Materials and Methods. B, Cytoplasmic and nuclear extracts of IL-7-expanded pro-B/pre-B cells were run in Western blot. The titration was 2, 4, and 8 μg of cell lysate per lane. E47, E12, and β-actin were detected with specific Abs (see Materials and Methods). Y, Young; O, old.