Masami Nakayama

HDAC2 controls IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions.

We previously reported that histone deacetylase‐2 (HDAC2) controls the amount of IgM H‐chain at the steps of transcription of its gene and alternative processing of its pre‐mRNA in DT40 cells. Here, we showed not only that the HDAC2‐deficiency caused repressions of gene expressions for HDAC7, EBF1, Pax5, Aiolos and Ikaros, and elevations of gene expressions for HDAC4, HDAC5, PCAF and E2A, but also that it caused altered acetylation levels of several Lys residues of core histones. Using gene targeting techniques, we generated three homozygous DT40 mutants: EBF1−/–, Aiolos−/– and E2A−/–, devoid of EBF1, Aiolos and E2A genes, respectively. Semiquantitative RT‐PCR analysis of the resultant mutants revealed not only that EBF1 and Aiolos down‐regulate expressions of IgM H‐ and L‐chain genes, but also that E2A up‐regulates expressions of these two genes. These results, together with others, indicate that HDAC2 controls indirectly expressions of IgM H‐ and L‐chain genes through opposite transcriptional regulations of EBF1, Pax5, Aiolos plus Ikaros and E2A genes.

Lacking of Aiolos accelerates pre-mature B cell apoptosis mediated by BCR signaling through elevation in cytochrome c release.

Antigen binding to B cell receptor (BCR) of pre-mature B lymphocytes leads to their apoptosis, while binding to BCR of mature B lymphocytes induces their activation and proliferation. The former binding is believed to be a mechanism so as to exclude B cell clones leading to protection from auto-immune diseases. Cross-linking of BCR of pre-mature B cells, including chicken DT40 cells, with anti-immunoglobulin antibody induces their apoptosis. The PMA/ionomycin treatments, which mimic BCR stimulation, are used to study intracellular signal transduction of B lymphocytes. Here, by analyzing the Aiolos-deficient DT40 cell line, Aiolos(-/-), we reveal that the lack of Aiolos accelerates apoptosis of DT40 cells mediated by BCR signaling. Moreover, the Aiolos-deficiency and BCR signaling cooperatively control this apoptosis through dramatically elevated cytochrome c release from mitochondria to cytosol and elevated caspase (caspase-3, 8 and 9) activities, resulting in drastically diminished amounts of ICAD followed by increased DNA fragmentation. Re-expression study reveals that the shorter isoform of Aiolos (Aio-2) controls PMA/ionomycin-mediated apoptosis via up-regulation and down-regulation of the PKCdelta and bak genes, respectively. These findings could be a powerful trigger to resolve molecular mechanisms of negative selection of B lymphocytes and also auto-immune diseases.

Participation of histones, histone modifying enzymes and histone chaperones in vertebrate cell functions.

Alterations in the chromatin structure are essential for easy accesses to chromosomal DNA. Such architectural alterations can be achieved by four means: (i) variants of histone subtypes, (ii) chromatin remodeling, (iii) post-translational modification, and (iv) chromatin assembly. This chapter discusses mainly on the first, third and fourth mechanisms, and especially on the acetylation of core histones, one of the third mechanisms. Taking the advantage of the gene targeting technique, we systematically established numerous mutant DT40 cell lines, each lacking particular gene, of interest such that encoding histones, histone deacetylases (HDACs), acetyltransferases (HATs) and chaperones, etc. Every subtype member of the histone gene family is capable of compensating the loss of others to maintain the mRNA level of each histone subtype, and most of histone variants are involved positively or negatively in the transcription regulation of particular genes. Regarding HDACs, HDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and HDAC-3 is indispensable for cell viability. Concerning HATs, GCN5 has tremendous impact on growth kinetics by preferentially acting as a supervisor in the normal cell cycle progression. The distinct participatory roles of the N-terminal and C-terminal halves of HIRA, one of histone chaperones, in both cell growth and transcription regulations of cell cycle-related genes, have also been highlighted. Therefore, the gene targeting technique in the DT40 cell line can be used as a powerful tool for the functional analysis of histones, histone modifying enzymes and histone chaperones relevant to chromatin biology.

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

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 2 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.

GCN5 is essential for IRF‐4 gene expression followed by transcriptional activation of Blimp‐1 in immature B cells

During B‐cell differentiation, the gene expression of B‐cell differentiation–related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5−/−, generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF‐4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp‐1 and IRF‐4. The ectopic expression of Blimp‐1 and IRF‐4 suggests that IRF‐4, but not Blimp‐1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF‐4 gene around its 5′‐flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF‐4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF‐4 gene expression, followed by transcriptional activation of Blimp‐1, and plays a key role in epigenetic regulation of B‐cell differentiation.

Lack of GCN5 remarkably enhances the resistance against prolonged endoplasmic reticulum stress-induced apoptosis through up-regulation of Bcl-2 gene expression

The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression.

GCN5 is involved in regulation of immunoglobulin heavy chain gene expression in immature B cells

GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency down-regulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions.

Possible involvement of Helios in controlling the immature B cell functions via transcriptional regulation of protein kinase Cs

The transcription factor Ikaros family consists of five zinc-finger proteins: Ikaros, Aiolos, Helios, Eos and Pegasus; these proteins except Pegasus are essential for development and differentiation of lymphocytes. However, in B lymphocytes, the physiological role of Helios remains to be elucidated yet, because its expression level is very low. Here, we generated the Helios-deficient DT40 cells, Helios (-/-), and showed that the Helios-deficiency caused significant increases in transcriptions of four protein kinase Cs (PKCs); PKC-δ, PKC-ε, PKC-η and PKC-ζ, whereas their expressions were drastically down-regulated in the Aiolos-deficient DT40 cells, Aiolos (-/-). In addition, Helios (-/-) was remarkably resistant against phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment, which mimics the B cell receptor (BCR)-mediated stimulation. In the presence of PMA/ionomycin, their viability was remarkably higher than that of DT40, and their DNA fragmentation was less severe than that of DT40 in the opposite manner for the Aiolos-deficiency. The resistance against the PMA/ionomycin-induced apoptosis of Helios (-/-) was sensitive to Rottlerin but not to Go6976. In addition, the Helios-deficiency caused remarkable up-regulation of the Rottlerin-sensitive superoxide (O2 (-))-generating activity. These data suggest that Helios may contribute to the regulation of the BCR-mediated apoptosis and O2 (-)-generating activity, via transcriptional regulation of these four PKCs (especially PKC-δ) in immature B lymphocytes. Together with previous data, our findings may significantly help in the understanding of the B lymphocyte-specific expressions of PKC genes and molecular mechanisms of both the BCR-mediated apoptosis involved in negative selection and the O2 (-)-generating system in immature B lymphocytes.

Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

SummaryNon-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.

Paired box gene 5 isoforms A and B have different functions in transcriptional regulation of B cell development-related genes in immature B cells.

The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5‐deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development‐related genes (activation‐induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma‐6, early B cell factor 1, origin binding factor‐1 genes), transcriptions of which are remarkably down‐regulated by Pax5‐deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.