E2A participates in a fine control of pre-mature B-cell apoptosis mediated by B-cell receptor signaling via transcriptional regulation of survivin, IAP2 and caspase-8 genes Kenji Toyonaga1,2,*, Hidehiko Kikuchi1,3,*, Koki Yamashita1, Masami Nakayama1, Kazuo Chijiiwa2 and Tatsuo Nakayama1,3 Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, Japan Section of Surgical Oncology and Regulation of Organ Function, Department of Medical Science, Miyazaki Medical College, University of Miyazaki, Japan Department of Life Science, Frontier Science Research Center, University of Miyazaki, Japan Keywords apoptosis; B cell; caspase; E2A; survivin Correspondence T Nakayama, Department of Life Science, Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Fax: +81 985 85 6503 Tel: +81 985 85 3127 E-mail: tnakayam@med.miyazaki-u.ac.jp *These authors contributed equally to this work (Received May 2008, revised 16 December 2008, accepted 24 December 2008) doi:10.1111/j.1742-4658.2009.06881.x Antigen binding to the B-cell receptor (BCR) of pre-mature B lymphocytes induces their apoptotic cell death, but binding to the BCR of mature B lymphocytes triggers activation and proliferation Binding to pre-mature B lymphocytes is thought not only to function as a mechanism to exclude B-cell clones that possess the ability to react with self-antigen, but also to act as a defense mechanism in auto-immune diseases Cross-linking of BCR of pre-mature B-cell lines, including the chicken DT40 cell line, with anti-immunoglobulin IgG induces apoptotic cell death Treatment with phorbol 12-myristate 13-acetate/ionomycin, which mimics BCR stimulation, is used to study intracellular signal transduction of B lymphocytes Here, by analyzing the E2A-deficient DT40 cell line, E2A)/), we show that E2A deficiency prevents certain levels of apoptotic cell death mediated by BCR signaling In addition, E2A deficiency-linked BCR signaling controls the mimicked pre-mature B-cell apoptosis by PMA/ionomycin through elevated survivin plus inhibitor of apoptosis levels, and reduced caspase-3 and caspase-8 activities, resulting in increased amounts of ICAD (inhibitor of caspase-activated DNase), compared with those in the presence of E2A, followed by reduction of DNA fragmentation These findings will contribute to the resolution of molecular mechanisms of negative selection of B cells and also auto-immune diseases It is widely known that B lymphocytes are essential to immune responses in health and disease, and maintain homeostasis by balancing cell viability and cell death [1] The molecular mechanisms of B-lymphocyte development have mostly been studied in mammalian bone marrow and/or peripheral lymphoid tissue (e.g spleen) Their development requires not only controlled lineageand locus-specific immunoglobulin gene recombination, establishing unique antigen specificity of the B lymphocytes, but also developmental stage-specific gene expression participating in lymphoid cell proliferation and synthesis of immune mediators [1–3] In addition, various factors and/or signals control various aspects of the normal development of B lymphocytes and function of the immune system Such developmental activities require numerous transcription factors, i.e the Abbreviations AIF, apoptosis-inducing factor; AKT, acutely transforming retrovirus AKT8 in rodent T cell lymphoma; BCR, B-cell receptor; CAD, caspaseactivated DNase; EBF, early B-cell factor; FACS, fluorescence-activated cell sorter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATA-3, GATA binding protein-3; HAT, histone acetyltransferase; HDAC, histone deacetylase; IAP, inhibitor of apoptosis; PARP, poly(ADPribose)polymerase; PCAF, p300/CBP-associated factor; PMA, phorbol 12-myristate 13-acetate 1418 FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS K Toyonaga et al basic helix-loop-helix transcription factors E2A, early B cell factor (EBF), GATA-binding protein-3 (GATA3), Pax5, PU.1, Ikaros and Aiolos, etc [2–4] Their importance in B-cell development has been established by knockout experiments on mouse hematopoietic stem cells, in which all these functions are associated with cessation of early stages of B-cell differentiation [3,5] However, the expression of these factors is sustained throughout development of lymphocytes in normal mice after the observed block in their knockout mice [6] Thus, their physiological functions beyond developmental arrest remain poorly understood Among these factors, E2A has been directly implicated in transcriptional regulation of several B lineagespecific genes, and has been shown to be essential for Ig H- and L-chain recombination [5] Moreover, E2A is required to initiate the expression of some B lineagespecific genes such as EBF, mb-1 and B29, but not to maintain expression of these genes [5] In general, E2A has also been shown to promote proliferation and survival of various cell types [5,7] Recently, it was revealed that E2A might be a key regulator of apoptosis versus proliferation in lymphoid cells, in addition to its B lineage-determining function [8–11] E2A-deficient mice develop T cell-derived lymphoma, and enforced expression of an inhibitor of differentiation proteins that indirectly inhibit E2A function [12,13] In addition, ectopic expression of E47 or E12 induced apoptotic cell death in thymic lymphoma cells derived from E2A-deficient mice [14] These results indicate that E2A might act as a tumor suppressor It is also accepted that B lymphocytes are susceptible to receptor- and mitochondria-initiated cell death at various stages of peripheral differentiation and during immune responses [1,15,16] Recent genetic evidence has contributed to understanding of the BCR-dependent survival mechanism of mature B cells [17,18] It is also known that E2A plays important roles in the apoptosis of B lymphocytes [9–11] In addition, Id3 protein, an E protein antagonist, induces growth arrest and apoptosis in B-lymphocyte progenitor cells [19] However, the impact of B cell-specific factors, including E2A, on the BCR-mediated apoptosis of pre-mature B cells remains unclear, when apoptosis is triggered by antigen stimulation In order to better understand the roles of histone acetyltransferases (HATs), histone deacetylases (HDACs) and B cell-specific transcription factors in B-cell functions, we have systematically generated various homozygous mutants, including HDAC2)/), GCN5)/), Aiolos)/) and E2A)/), using gene-targeting techniques on the DT40 cell line, which was established from chicken pre-mature B lymphocytes [20] Our Fine control of pre-mature B-cell apoptosis by E2A results show that HDAC2 controls the amount of IgM H-chain at two stages: transcription of its gene and alternative processing of its pre-mRNA [21] Recently, we revealed not only that HDAC2 upregulates gene expression of EBF1, Pax5, Aiolos, Ikaros and HDAC7, and down-regulates those of E2A, p300/CBP-associated factor (PCAF), HDAC4 and HDAC5, but also that E2A upregulates expression of IgM H- and L-chain genes, but downregulates Aiolos, but Aiolos, EBF1, Pax5, and Ikaros downregulate expression of these two genes [22] These results, together with others, indicate that HDAC2 indirectly controls the expression of IgM H- and L-chain genes, through upregulated transcriptional regulation of EBF1, Pax5, Aiolos and Ikaros, and downregulated transcriptional regulation of E2A Moreover, our results indicated that GCN5 functions as a supervisor in normal cell-cycle progression, with comprehensive control over the expression of several cell cycle-related genes, as well as apoptosis-related genes, probably through alterations in the chromatin structure, indicated by the changing acetylation status of core histones surrounding these widely distributed genes [23] Recently, we showed that GCN5 and BCR signaling collaborate to induce apoptotic cell death of the DT40 cell line, through depletion of ICAD [inhibitor of caspase-activated DNase (CAD)] and inhibitor of apoptosis (IAP2), and activation of caspase activities [24] Based on these results, in this study, as a first step in elucidating the participation of B cell-specific factors in pre-mature B-cell apoptosis mediated by BCR stimulation by analyzing the E2A-deficient DT40 mutant E2A)/), we clarified the impact of E2A on apoptotic cell death of the DT40 cell line, and show that E2A is involved in fine control of pre-mature B-cell apoptosis mediated by BCR signaling, via transcriptional regulation of survivin, IAP2 and caspase-8 genes Results Insignificant influence of E2A deficiency on gene expression of apoptosis-related factors To assess the influence of E2A deficiency on gene expression of apoptosis- and BCR signaling-related factors, and other factors, we performed semi-quantitative RT-PCR on total RNAs prepared from DT40 and three independent E2A)/) clones (Fig 1) E2A deficiency did not have a significant influence on transcription of most of these genes, except for survivin (to approximately 200%), PKCa (to approximately 60%), PKCg (to approximately 40%), PKCl (to approximately 40%) and PKCf (to approximately 160%) In addition, we performed immunoblot analyses to assess the influence FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS 1419 Fine control of pre-mature B-cell apoptosis by E2A K Toyonaga et al Fig Effect of E2A deficiency on gene expression of apoptosis-related factors, caspases, caspase-regulating factors, CAD/ICAD and PKCs Total RNAs were extracted from DT40 and three independent E2A)/) clones (1–3), and mRNA levels were determined by semi-quantitative RT-PCR using appropriate primers The chicken GAPDH gene was used as an internal control The numbers under the panels indicate the number of cycles used for PCR of E2A deficiency on the amounts of proteins whose mRNA levels were altered as noted above Consistent with the results on mRNA levels, the protein levels for survivin and PKCf were increased in E2A)/) and that of PKCg was decreased (Fig S1); PKCa and PKCl could not be detected using the available antibodies On the other hand, transcription of various genes encoding membrane-proximal factors, NF-jBs, transcription factors and B cell-related factors, amongst others, was not altered in the E2A-deficient mutants (data not shown) These insignificant effects of E2A deficiency on the expression of numerous genes probably resulted in no changes in the amount of apoptotic cells, as discussed below (see Fig 2) Moderate resistance to apoptosis is induced by PMA/ionomycin in E2A-/We examined the influences of phorbol 12-myristate 13-acetate (PMA)/ionomycin and etoposide on 1420 cell-cycle progression and proliferation of DT40 and E2A)/) cells Cells cultured in the presence of PMA/ ionomycin for 24 h or etoposide for h were analyzed by fluorescence-activated cell sorter (FACS) after staining with propidium iodide (Fig 2A) As expected, etoposide treatment caused apoptotic cell death for the two cell lines at the same level because the drug inhibits the topoisomerase-2 activity that is essential for DNA replication Although PMA/ionomycin treatment of DT40 cells resulted in cell-cycle distributions that were quite different from those in the case of the etoposide treatment, it did induce apoptosis, and these findings agree with those reported previously [24] On the other hand, although PMA/ionomycin treatment of E2A)/) did not alter the cell-cycle distribution pattern up to 24 h (cell growth was slightly delayed thereafter), the depletion of E2A slightly prevented apoptotic cell death even in the presence of PMA/ ionomycin To confirm these findings, we examined the effects of PMA/ionomycin treatment on the viability FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS K Toyonaga et al A Fine control of pre-mature B-cell apoptosis by E2A B C Fig Analyses of apoptosis in E2A)/) (A) Effects of PMA/ionomycin and etoposide treatments on cell-cycle distributions of DT40 and E2A)/) DT40 and E2A)/) cells treated with etoposide (10 lgỈmL)1) for h, or with PMA (10 ngỈmL)1) plus ionomycin (1 lM) for 24 h, were processed for DNA content analysis by propidium iodide staining Nuclei were analyzed by flow cytometry (FACSCalibur, Becton Dickinson and Company, Franklin Lakes, NJ, USA), and data for DT40 and E2A)/) (clone 1) were plotted on linear histograms as relative cell number (y axis) against red fluorescence intensity (x axis) The percentages of the various cell-cycle phases (sub-G1, G1, S and G2/M) for for DT40 and E2A)/) (clone 1), together with those of two other E2A)/) clones (2 and 3) are indicated in the table (B) Sensitivity of DT40 (circles) and E2A)/) (squares, triangles and diamonds) to PMA/ionomycin-mediated apoptotic cell death Cells were resuspended in DMEM containing 10% v/v fetal bovine serum, and treated with (filled symbols) or without (open symbols) 10 ngỈmL)1 PMA plus lM ionomycin at 37 °C for up to 48 h Viable cells were counted by the trypan blue dye exclusion method Data represent the mean of two separate experiments, and error bars indicate the standard deviation (C) Morphology of DT40 and E2A)/) (clone 1) cells treated with PMA/ionomycin Cells were cultured for 24 h without (no treatment) or with PMA/ionomycin (PMA/ionomycin), and their nucleus forms were analyzed by microscopy of DT40 and E2A)/) (Fig 2B) As expected, the viability of the two cell lines did not differ in the absence of PMA/ionomycin However, in the presence of PMA and ionomycin, the viability of E2A)/) (approximately 50% at 48 h) was slightly higher than that of DT40, which was dramatically reduced (approximately 20% by 48 h) We undertook a comparative analysis of changes in the morphological structure of nucleus as an effect of PMA/ionomycin treatment in DT40 and E2A)/) (Fig 2C) Nuclear fragmentation, another characteristic of apoptosis, was partially hindered in E2A)/), but it was clearly detected for DT40 in the presence of PMA/ionomycin These results show that E2A deficiency leads to blockage of the induced apoptotic cell death that is seen in the DT40 cell line when treated with PMA/ionomycin Influence of depletion of B cell-specific transcription factors, HDACs and HATs on resistance to apoptosis induced by PMA/ionomycin treatment Using gene-targeting techniques, we systematically generated several homozygous DT40 mutants that lacked genes encoding B cell-specific transcription factors, HDACs or HATs [20,22] Our previous results revealed that GCN5 and PMA/ionomycin treatment FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS 1421 Fine control of pre-mature B-cell apoptosis by E2A K Toyonaga et al Table Influences of depletion of B cell-specific transcription factors, HDACs and HATs on resistance to apoptosis induced by PMA/ionomycin treatment Mutants B cell-specific factors E2A)/) Aiolos)/) EBF)/) Helios)/) Pax5)/) HDACs HDAC1)/) HDAC2)/) HDAC7)/) SIRT1)/) SIRT2)/) HATs GCN5)/) PCAF)/) HAT1)/) MORF)/) MOZ)/) MOZ)/)/MORF)/) Reference Resistance for PMA/ionomycin [22] [22] [22] Unpublished data [22] › fl fi fi fi [21] [21] [23] [31] [31] fi › fi fi fi [23] [23] [32] Unpublished data Unpublished data Unpublished data › fi fi fi fi fi cooperatively induce apoptotic cell death in the DT40 cell line [24] As a first step in elucidating the participation of E2A in apoptosis of DT40 cells, we examined the effects of PMA/ionomycin treatment for these homozygous DT40 mutants by FACS after staining with propidium iodide and/or determination of cell viability Detailed information on the generation of mutants lacking Helios, Pax5, MORF, MOZ and MOZ/MORF will be shown elsewhere As shown in Table 1, depletion of HDAC2, as well as of E2A and GCN5, prevented the apoptosis induced by PMA/ionomycin treatment, but depletion of Aiolos promoted such apoptosis No changes were detected in the remaining single mutants lacking EBF, Helios, Pax5, HDAC1, HDAC7, SIRT1, SIRT2, PCAF, HAT1, MORF or MOZ, or in a double knockout mutant lacking MOZ and MORF These findings suggest that, among the B cell-specific transcription factors, HDACs and HATs examined, E2A, Aiolos, HDAC2 and GCN5 preferentially participate in control of the apoptotic cell death induced by PMA/ionomycin treatment of the DT40 cell line Up- and downregulation of expression of the E2A or Aiolos genes by PMA/ionomycin treatment To determine whether or not expression of the B cell-specific transcription factors, HDACs and HATs 1422 mentioned above is influenced by PMA/ionomycin treatment, DT40 and E2A)/) were cultured in the presence of PMA/ionomycin, and RT-PCR was performed (Fig and Fig S2) E2A deficiency dramatically decreased the mRNA level of Aiolos (to approximately 30%), but did not have any effect on transcripts of the remaining B cell-specific transcription factors, or HDACs and HATs On the other hand, PMA/ionomycin treatment dramatically altered gene expression of E2A in DT40 (to approximately 320% by 24 h) and that of Aiolos in both DT40 and E2A)/) (to less than 10% by 24 h) However, in the two cell lines, the treatment did not have significant effects on mRNA levels of HDAC1, HDAC2, SIRT2, HAT1 and MORF, and slightly distinct but almost similar effects on mRNA levels of EBF, Pax5, HDAC7, SIRT1, GCN5, PCAF and MOZ These findings, together with those shown in Table 1, indicate not only that HDAC2 and GCN5 are necessary for control of the apoptosis of the DT40 cell line mediated by PMA/ionomycin treatment, but also that their own transcription is not influenced by PMA/ionomycin Therefore, the apoptotic cell death of DT40 mediated by PMA/ionomycin treatment must be under the control of the elevated or decreased amounts of E2A or Aiolos Upregulation of survivin and IAP2 gene expression and no effect on caspase-8 gene expression by PMA/ionomycin treatment in E2A-/To further clarify the molecular mechanism linked to the apoptotic induction of the DT40 cell line cooperatively mediated by E2A and BCR signaling, DT40 and E2A)/) were cultured in the presence of PMA/ionomycin, and RT-PCR was performed for various factors (Fig and Fig S2) E2A deficiency did not alter the gene expression of the apoptosis-related factors bcl-2, bcl-xL, bak, Apaf-1, cytochrome c, acutely transforming retrovirus AKT8 in rodent T cell lymphoma (AKT), apoptosis-inducing factor (AIF) and poly (ADP-ribose)polymerase (PARP) (also shown in Fig 1) The PMA/ionomycin treatment did not have a significant influence on the mRNA levels of bcl-2, bak, Apaf-1, cytochrome c, AKT and AIF, and had slightly distinct but almost similar effects on the mRNA levels of bcl-xL and PARP in both DT40 and E2A)/) Similarly, depletion of E2A showed no effects on gene expression of CAD and ICAD (also shown in Fig 1) PMA/ionomycin treatment showed similar effects on the expression of CAD and ICAD genes in both DT40 and E2A)/), i.e the CAD mRNA level decreased by h but thereafter increased to the control level by 24 h, and the ICAD mRNA level FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS K Toyonaga et al Fine control of pre-mature B-cell apoptosis by E2A Fig Effects of PMA/ionomycin treatment on gene expression of B cell-specific factors, HDACs, HATs, apoptosis-related factors, CAD/ ICAD, caspases and caspase-regulating factors Total RNAs were extracted from PMA/ionomycin-treated DT40 and E2A)/) (clone 1) at indicated times up to 24 h, and then the mRNA levels of appropriate genes were determined by RT-PCR The chicken GAPDH gene was used as a control decreased by h and thereafter remained unchanged at 24 h in the presence of PMA/ionomycin These findings indicate that E2A and BCR stimulation have no effects on gene expression of CAD and ICAD, in contrast to the effects of GCN5 and BCR stimulation [24] With regard to caspases, E2A depletion showed no effects on expression of caspase-3, caspase-6, caspase8, caspase-9 and caspase-10 genes (also shown in Fig 1) On the other hand, in both DT40 and E2A)/), the caspase-6 mRNA level was decreased gradually by PMA/ionomycin treatment by 24 h, and the caspase-10 mRNA level was increased by h and thereafter decreased dramatically by 24 h Expression of caspase-3 and caspase-9 remained unchanged in the presence of PMA/ionomycin Interestingly, in DT40, PMA/ionomycin treatment increased the caspase-8 mRNA level by h (to approximately 160%) and this level remained unchanged at 24 h, but the treatment showed no change in the transcript level of caspase-8 in E2A)/) These findings indicate that expression of most caspase genes is not much influenced by either E2A or BCR stimulation, except that of caspase-8 With regard to caspase-regulating factors, depletion of E2A increased transcription of the survivin gene (to approximately 220%), but did not have a significant effect on expression of the FLIP, IAP1, IAP2 and Smac genes However, PMA/ionomycin treatment had distinct effects on expression of these caspase inhibitors The IAP1 or Smac mRNA levels increased or decreased slightly by h and thereafter remained unchanged in both DT40 and E2A)/) The FLIP mRNA level was slightly decreased at h (to approximately 60%) and thereafter increased to the control level in DT40, but remained unchanged in E2A)/) FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS 1423 Fine control of pre-mature B-cell apoptosis by E2A K Toyonaga et al Interestingly, whereas the IAP2 mRNA level in DT40 was significantly decreased by PMA/ionomycin treatment at h (to approximately 30%) and thereafter remained unchanged, the transcript level in E2A)/) was unchanged at h, and thereafter decreased by 24 h (to approximately 20%) Furthermore, in E2A)/), the survivin mRNA level was maintained at a high level (approximately 220%) when treated with PMA and ionomycin for h, and thereafter decreased to slightly higher level than that in DT40; the mRNA level was not influenced by the drug treatment in DT40 These findings indicate not only that E2A downregulates transcription of the survivin gene and has no effects on that of FLIP, IAP1, IAP2 or Smac, but also that BCR stimulation and E2A cooperatively control expression of FLIP, IAP2 and survivin genes Resistance to PMA/ionomycin-mediated apoptosis of E2A-/- is brought about by increased amounts of survivin and IAP2, and reduced activity of caspase-3 Next we examined the effect of PMA/ionomycin treatment on cellular protein levels of survivin, IAP2 and ICAD, which are proximal factors controlling CAD activity for DNA fragmentation, by immunoblotting using their specific antibodies (Fig 4A) E2A deficiency increased the protein levels of survivin (to approximately 190%), but had no effect on those of IAP2 and ICAD Consistent with previous results [24], PMA/ionomycin treatment in DT40 dramatically decreased the protein levels of IAP2 and ICAD (to approximately 40% and less than 10%) by 24 h, but had a moderate influence on that of survivin (approximately 60% at 24 h) Therefore, in DT40 cells treated with PMA/ionomycin, the time courses of alterations in the protein and mRNA levels of survivin were virtually similar, and the gradual reductions in protein levels of IAP2 plus ICAD compared with acute decreases (unchanged thereafter) of their mRNA levels agreed with previous results [24] On the other hand, in E2A)/), PMA/ionomycin treatment did not influence the protein level of survivin by h and thereafter it decreased gradually PMA/ionomycin treatment did not change the protein level of IAP2 Further, the PMA/ionomycin treatment showed a moderate reduction in the protein level of ICAD (to approximately 40%) by 24 h in E2A)/), i.e the rate of decrease in the amount of ICAD in the mutant was slower than that in DT40 Thus, in E2A)/), in the presence of PMA/ionomycin, the time courses of alterations in the mRNA and protein levels of survivin were virtually similar up to 24 h, but the findings that the protein levels of IAP2 or ICAD were increased or 1424 slightly decreased by 24 h did not agree with the findings that the mRNA levels of IAP2 and ICAD were reduced slowly by 24 h or quickly by h, respectively To resolve this discrepancy between the results regarding the mRNA and protein levels of ICAD (and also IAP2), we next examined the effects of PMA/ ionomycin treatment on caspase activities (Fig 4B) Interestingly, in E2A)/), PMA/ionomycin treatment showed slightly decreased activities of caspase-3 (to approximately 50% by 16 h), caspase-8 (to approximately 70% by 16 h) and caspase-9 (to approximately 70% by 16 h), compared with those in DT40, probably due to a balance of the amounts of each of the three caspases and the inhibitors survivin and IAP2 (and also FLIP and IAP1) The slightly decreased caspase-8 activity mediated by PMA/ionomycin treatment in E2A)/) may have resulted from the balanced mRNA (and probably protein) levels of caspase-8, which lead to activation of pro-caspase-9, causing formation of the active form of caspase-3, and of the inhibitors FLIP (for caspase-8), survivin (for caspase-9) and IAP2 (for caspase-3) [25] In E2A)/), the decreased activity of caspase-3 mediated by PMA/ionomycin treatment must depend on both the reduced amount of activated caspase-3 itself as a result of decreased caspase-8 (and probably caspase-9) activity, and the elevated protein (and also mRNA) levels of the inhibitors survivin and IAP2 As a result, the slow diminution of the protein level of ICAD by PMA/ionomycin treatment in E2A)/) (Fig 4A) must be due to its slight degradation mediated by suppressed caspase-3 activity, although the ICAD mRNA level was decreased when exposed for h but thereafter remained unchanged, as did that in DT40 (Fig and Fig S2) Because the alterations in the mRNA level of CAD were the same in both DT40 and E2A)/) (Fig and Fig S2), and its protein could not be detected by the available antibodies to assess CAD activity, we examined the effects of PMA/ionomycin treatment on DNA fragmentation, a typical result of CAD activity (Fig 4C) The DNA fragmentation was found to be more moderate for E2A)/) than that observed for DT40, even in the presence of PMA/ionomycin up to 24 h These results indicate that the CAD activity in E2A)/) is suppressed by the moderately reduced amount of its inhibitor ICAD compared with that in DT40, resulted in decreased DNA fragmentation, a characteristic of apoptosis Discussion In recent years, numerous studies have been performed to determine the physiological target genes of E2A FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS K Toyonaga et al Fine control of pre-mature B-cell apoptosis by E2A A B C Fig Analyses of mechanisms of the resistance to PMA/ionomycin-mediated apoptosis in E2A)/) (A) Effects of PMA/ionomycin treatment on protein levels of survivin, IAP2 and ICAD Whole proteins were isolated from PMA/ionomycin-treated DT40 and E2A)/) at indicated times up to 24 h, and subjected to SDS–PAGE followed by immunoblotting Antibody binding was detected using secondary antibodies conjugated to horseradish peroxidase, and then data analysis was performed using a luminescent image analyzer Left panel: typical immunoblot pattern (DT40 and E2A)/) clone 1) b-actin was used as a control The apparent molecular masses of marker proteins are indicated Right panel: time courses of protein levels for survivin, IAP2 and ICAD after treatment with PMA/ionomycin in DT40 (circles) and three E2A)/) clones (1–3) (squares, triangles and diamonds) Data are expressed as percentages of the control (DT40 at h) (B) Effects of PMA/ionomycin treatment on caspase activities Cell lysates were prepared from PMA/ionomycin-treated DT40 (circles) and E2A)/) clones (1–3) (squares, triangles and diamonds) at indicated times up to 16 h, and then caspase activity assays were performed using appropriate caspase assay kits Absorbance at 405 nm was measured to determine activities Data represent the mean of two separate experiments, and error bars indicate standard deviation (C) Effects of PMA/ionomycin treatment on DNA fragmentation in DT40 and E2A)/) DNA was isolated from DT40 and E2A)/) cells incubated for 0, 8, 16 and 24 h in the presence of PMA and ionomycin, and analyzed by 1.5% agarose gel electrophoresis The sizes of k-DNA digested with HindIII are indicated in kb Left panel: typical electrophoregram of DNA extracted from PMA/ionomycin-treated DT40 and E2A)/) (clone 1) Right panel: electrophoregram of DNA extracted from PMA/ionomycin-treated DT40 and three E2A)/) clones (1–3) at 16 h FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS 1425 Fine control of pre-mature B-cell apoptosis by E2A K Toyonaga et al E2A directly activates the EBF gene [26] and regulates expression of several genes, i.e k5, Rag-1, Vj1 and jo, that are involved in D-JH rearrangement, cell survival, Igj rearrangement, etc [26,27] E2A directly controls IgH gene expression, and is involved in repressing the Nfil3 and FGFR2 genes in pre-mature B lymphocytes [28] Thus, E2A is one of the most essential regulators at multiple stages of B-cell development In T lymphocytes, PLCc2, Cdk6, CD25, Tox, Gadd45a, Gadd45b, Gfi1, Gfi1b, Socs1, Socs3, Id2, Eto2, Xbp1 etc have been identified as novel E47 target genes using an E2Adeficient lymphoma cell line [29] Recently, we reported that apoptosis of the chicken DT40 cell line, a premature B-cell line, is cooperatively controlled by GCN5 and BCR stimulation via complex transcriptional regulation of a number of genes encoding BCR signaling-related factors, B cell-specific factors, transcription factors and apoptosis-related factors, indicating that both are necessary for apoptosis of DT40 cells [24] In DT40 cells, BCR signaling is transduced from BCR and membrane-proximal factors (Syk, BTK, BLNK and PLCc2, etc), via mainly PKCd, PKCe and PKCf, to NF-jBs (probably c-Rel and NFp50) This activated signal mediated by BCR signaling is probably transducted separately into two apoptotic pathways, i.e direct transduction of the signal into the CAD/ICAD system and transduction of the activated signal into the caspase cascade pathway However, understanding of the participation of most B cell-specific factors in the apoptotic process has remained elusive Lack of E2A partially prevents the apoptotic cell death seen in DT40 cells treated with PMA/ionomycin, which mimics BCR stimulation (Fig 2); such apoptosis is completely prevented by either GCN5 deficiency [24] or HDAC2 deficiency, and is significantly accelerated by Aiolos deficiency (Table 1) By analyzing E2A)/), we revealed that E2A upregulates the expression of PKCa, PKCg and PKCl genes, and downregulates the expression of survivin and PKCf genes, among the numerous factors examined (Fig and unpublished data) PMA/ ionomycin treatment increased expression of the E2A gene and dramatically suppressed that of the Aiolos gene in DT40, but in E2A)/) had no effects or similar effects on the expression of other disrupted genes (Fig 3A) and genes encoding B cell-specific factors, HDACs and HATs (our unpublished data) These results suggest not only that, among the B cell-specific factors tested, E2A or Aiolos participates preferentially in suppression or acceleration of apoptosis of the DT40 cell line, but also that, among the HAT and HDAC families tested, gene expression of GCN5 and HDAC2, which are essential for apoptotic cell death, are not influenced directly by PMA/ionomycin treatment On 1426 the other hand, in E2A)/), the detected protein level of Aiolos, which was already suppressed to a very low level, is further reduced by the treatment, resulting in suppression of apoptotic cell death compared with that of DT40 The alterations in expression of E2A and Aiolos (and probably others) in DT40 when exposed to PMA/ionomycin accompany altered expression of various genes encoding apoptosis-related factors, caspases, caspase-regulating factors and CAD/ICAD (Fig and Fig S2), and almost all of these results agreed with those in a previous report [24] In E2A)/), the altered expression of Aiolos (and probably others) in the presence of PMA/ionomycin results in (and/or accompanies) slightly different effects on expression of the genes mentioned above, i.e the influences of PMA/ionomycin treatment on expression of the survivin and IAP2 (and probably FLIP) genes were more moderate in the mutant than in DT40, and no effect was observed on expression of caspase-8 However, in DT40, the PMA/ionomycin-induced alterations in gene expression of various B cell-specific factors, HDACs, HATs, apoptosis-related factors, caspases, caspase-regulating factors and CAD/ICAD led to changes in the activities of caspase-3, caspase-8 and caspase-9, and in the protein levels of IAP2 and ICAD (Fig 4A,B), consistent with previous results [24] Interestingly, in E2A)/), the noticeable alterations in gene expression of survivin and IAP2 accompanied by unchanged gene expression of caspase-8 (and also various B cell-specific factors, HDACs, HATs, apoptosis-related factors, caspases, caspase-regulating factors and CAD/ICAD) mediated by PMA/ionomycin results in suppression of activities of caspase-3, caspase-8 and caspase-9, and alterations in the protein levels of survivin, IAP2 and ICAD (and probably FLIP) (Fig 4A,B) Finally, in E2A)/), the slightly suppressed degradation of ICAD molecules as an effect of reduced caspase-3 activity reduces CAD activity, leading to moderate fragmentation of DNA molecules (Fig 4C) Thus, progress towards apoptotic cell death in E2A)/) is suppressed by collaboration of both BCR signaling and E2A depletion, mainly via moderate changes in amounts of the inhibitors survivin, IAP2 and ICAD (and probably FLIP) Thus, E2A is involved in fine control of pre-mature B-cell apoptosis mediated by BCR signaling via transcriptional regulation of survivin, IAP2, FLIP and caspase-8 genes The observations in this study regarding the participation in apoptosis of Aiolos and HDAC2, which are now being further studied by us, as well as that of GCN5, amongst others, will be useful in elucidating not only the linkage between BCR signaling and FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS K Toyonaga et al apoptosis cascades in pre-mature B-cell lines, but also the molecular mechanism of negative selection or development of B lymphocytes through cross-talk among B-cell signaling, B cell-specific transcriptional regulation and epigenetic chromatin topology alterations These results, combined with other findings obtained in the future, may contribute to clinical understanding of auto-immune diseases and B-cell lymphomas Fine control of pre-mature B-cell apoptosis by E2A Immunoblotting Cells were treated with 10% trichroloacetic acid, collected by centrifugation 20 000 g for at °C, dissolved in 0.5 m Tris/HCl (pH 6.8) containing 2.5% SDS, 10% glycerol and 5% 2-mercaptoethanol, and heated at 100 °C for Immunoblotting was performed as described previously [24] b-actin was used as a control Acknowledgements Experimental procedures Materials PMA was purchased from Calbiochem (Darmstadt, Germany) and ionomycin was purchased from Sigma (St Louis, MO, USA) The antibodies used were anti-ICAD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anticIAP-2 (Chemicon, Temecula, CA, USA), horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin and horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dako Inc., Glostrup, Denmark) Cell cultures and apoptosis induction DT40 cells and all subclones were cultured essentially as described previously [24] Apoptosis was induced as follows: cells (2 · 106) in 10 mL of culture medium were incubated with 10 ngỈmL)1 PMA plus lm ionomycin or with 10 lgỈmL)1 etoposide at 37 °C Viable cells were counted by the trypan blue dye exclusion method Flow cytometric analyses, morphological analyses, the caspase activity assay and the DNA fragmentation assay were performed as described previously [24,30] Semi-quantitative RT-PCR Total RNAs were isolated from DT40 and its subclones Reverse transcription was performed using a first-strand DNA synthesis kit (Toyobo, Osaka, Japan) at 42 °C for 20 min, followed by heating at 99 °C for PCRs were performed as described previously [24] using sense primers and antisense primers synthesized according to the EST data deposited in GenBank for the appropriate genes, and listed in previous reports [22–24], except for SIRT1 (sense primer 5¢-CTGTTTTTACCACCAAATCG-3¢ and antisense primer 5¢-CAACTTGTTGCTTGTTGGAT-3¢) and SIRT2 (sense primer 5¢-ATGTCCCTCATGGGCTTCGG-3¢ and antisense primer 5¢-TCACGGCTCTTTGTCGTCCC-3¢) The chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control PCR products were subjected to 1.5% agarose gel electrophoresis, and analyzed using an LAS-1000plus luminescent image analyzer (Fujifilm, Tokyo, Japan) We thank Y Takami and H Suzuki for technical support and H K Barman for editorial reading of the manuscript This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan References Cancro MP (2005) B cells and aging: gauging the interplay of generative, selective, and homeostatic events Immunol Rev 205, 48–59 Singh H, Medina KL & Pongubala JMR (2005) Contingent gene regulatory networks and B cell fate specification Proc Natl Acad Sci USA 102, 4949–4953 Kee BL & Murre C (2001) Transcription factor regulation of B lineage commitment Curr Opin Immunol 13, 180–185 Riley RL, Van der Put E, King AM, Fransca D & Blomberg BB (2005) Deficient B lymphopoiesis in murine senescence: potential roles for 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Nishijima H, Sanematsu F, Shibahara K & Nakayama T (2006) Histone acetyltransferase is dispensable for replication-coupled chromatin assembly but contributes to recover DNA damage created following replication blockage in vertebrate cells Biochem Biophys Res Commun 345, 1547–1557 Supporting information The following supplementary material is available: Fig S1 Influences of E2A deficiency on protein levels of survivin, PKCg and PKCf Fig S2 Effects of the PMA/ionomycin treatment on gene expression of B cell-specific factors, HDACs, HATs, apoptosis-related factors, CAD/ICAD, caspases and caspase-regulating factors in two E2A)/)clones This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 1418–1428 ª 2009 The Authors Journal compilation ª 2009 FEBS ... show that E 2A is involved in fine control of pre-mature B-cell apoptosis mediated by BCR signaling, via transcriptional regulation of survivin, IAP2 and caspase-8 genes Results Insignificant in? ??uence... BCR signaling and E 2A depletion, mainly via moderate changes in amounts of the inhibitors survivin, IAP2 and ICAD (and probably FLIP) Thus, E 2A is involved in fine control of pre-mature B-cell apoptosis. .. apoptosis mediated by BCR signaling via transcriptional regulation of survivin, IAP2, FLIP and caspase-8 genes The observations in this study regarding the participation in apoptosis of Aiolos and HDAC2,