Department of Molecular and Developmental Biology

Summary

Our long-term goal is to understand the molecular mechanisms which coordinately regulate growth and differentiation of metazoan cells with emphasis on intracellular signal transduction, transcription and DNA replication. For this purpose, we are using systems ranging from E. coli, yeasts, frog, zebrafish, and culture cells of lymphocytic and hematopoietic lineages as well as animal models. The major research areas of interest are on: 1) transcriptional regulation of Th1 and Th2-specific cytokine genes in activated T cells, 2) roles of cytokines and their receptors in growth and differentiation, 3) regulation of DNA replication and cell cycle and 4) development of sensory organs. On the basis of these efforts, we intend to develop technologies to manipulate growth and differentiation of various stem cells with high fidelity, which is important for cell and gene therapy.

I. Regulation of lymphocyte differentiation and activation by transcription factors

CD4 naive precursor T cells are the population of CD4 positive T cells that have matured in thymus and have never been exposed to real antigens. They are characterized by low CD44 and high CD62L. Recognition of antigens by naive precursor T cells leads to extensive proliferation and production of cytokines. Among various cytokines, activated naive precursor T cells produce mainly IL-2. Concomitantly activated naive precursor T cells differentiate into one of the two T helper subsets, Th1 and Th2. Th1 and Th2 cells are characterized by the spectrum of cytokines they produce. Th1 cells are one of the important producers of interferon g (IFNg) and lymphotoxin (LT), while Th2 cells produce Interleukin (IL)-4, IL-5, IL-6, IL-10 and IL-13. Resting T helper subsets are characterized by high CD44 and low CD62L and they are designated as memory CD4 T cells. Th1 cytokine, such as IFNg is crucial for the activation of macrophages and cellular immune responses against intracellular pathogens. Th2 cytokines such as IL-4, IL-5 and IL-13 regulate B cell activation, antibody production and differentiation and activation of eosinophils. Thus, Th2 cells play important roles in protection against extracellular pathogens, especially helminth parasites. However, inappropriate activation of these T helper subsets can cause various immunological disorders. Many organ specific autoimmune diseases such as rheumatoid arthritis and multiple sclerosis are suggested to be triggered by the Th1 cells recognizing tissue-specific self antigens. Activation and expansion of Th2 cells against particular antigens are probably the most critical culprits of allergic responses such as asthma and allergic dermatitis.
The differentiation of T helper subsets requires DNA replication and cell proliferation. In addition, it is regulated by various parameters incurred by stimuli, such as the strength and nature of the signal through TCR, type of costimulatory signals, type of antigen presenting cells including dendritic cell subsets etc. Among various conditions, two cytokines are crucial to regulate the process. IL-12 produced by antigen presenting cells skew the differentiation process toward Th1 cells, while IL-4 is effective to induce Th2 cells. Our focus is the role of transcription factors such as STAT6, GATA3 and NFAT family proteins inTh2 differentiation pathway, with particular focus on their role in the remodeling of chromatin structures during induction of Th2 subset specific gene expression. Furthermore, we are studying the role of STAT6 in other tissues in allergic responses.

1. Chromatin remodeling of the mouse IL-4 and IL-13 loci in Th2 differentiation

Naofumi Takemoto1, Shoichiro Miyatake1, Yumiko Kamogawa1, Miho Nagoya, Sahori Namiki1, Naoko Arai2 and Ken-ichi Arai1
1CREST (Japan Science and Technology), 2Department of Immunology, DNAX Research Institute

Many cytokine genes are located on the human chromosome 5 and its syntenic region is found on the mouse chromosome 11. Three of the Th2 specific cytokine genes, IL-4, IL-13 and IL-5 are clustered on these chromosomes. IL-4 and IL-13 genes are only 12 kb apart, whereas IL-5 and IL-13 genes are separated by rad50 gene which is about 120 kb long. We identified three DNase I hypersensitive sites (HSS) in the intergenic region of IL-4 and IL-13 genes. Two of them, HSS1 and HSS2 are detected in both resting and activated Th2 cells but not in naive precursor cells and Th1 cells. The appearance of these Th2 specific HSS is not associated with the actual transcription of IL-4 or IL-13 genes, suggesting that these HSS reflect Th2 cell-specific chromatin structures. We could show that both Th2 differentiation and the induction of Th2-specific chromatin structures can be achieved by activation of STAT6 or exogenous expression of GATA3 even in the Th1 skewing condition. These data strongly suggest that GATA3 induced by STAT6 is the key regulator for Th2 specific chromatin remodeling. One putative GATA binding site was identified within the region containing HSS2. The binding of GATA3 to this site in vitro was shown by EMSA. Binding of GATA3 to this site in the intact chromatin requires further investigation. We also identified Th2-specific DNA binding complex which recognizes a putative AP-1 binding site within the region containing HSS1. Since AP-1 DNA binding activity can be detected in both Th1 and Th2 cells, this Th2 specific complex is different from the known AP-1 complexes. Identification of the functional domains in GATA3 has been carried out by introducing various mutant GATA3 into in vitro differentiating precursor T cells. We showed that one of the two zinc fingers, located closer to N terminus, is dispensable for the expression of IL-4 but not for IL-5. Th2 specific HSS are also induced by the GATA3 mutant lacking this finger. Since it was reported previously that this zinc finger as well as other fingers of GATA3 are required for its DNA binding activity, DNA binding activity of GATA3 may not be required for remodeling of the Th2 cell-specific chromatin structures. On the other hand, transcription of IL-5 may require GATA3 DNA binding activity. Further biochemical analyses of GATA3 and associating molecules are in progress to clarify the role of GATA3 in chromatin remodeling.

2. The role of STAT6 in Th2 differentiation and the pathology of airway hyperresponsiveness

Yumiko Kamogawa1, Shoichiro Miyatake1, Miho Nagoya, Sahori Namiki1, Naofumi Takemoto and Ken-ichi Arai1
1CREST (Japan Science and Technology)

IL-4 is a critical cytokine for the differentiation of Th2 cells which may trigger allergic responses. It has been shown that IL-4 and IL-13, Th2 specific cytokines, may play important roles in the pathology of asthma not only through induction of Th2 cells but also through directly affecting airway tissues such as airway epithelial cells and smooth muscle cells. STAT6 is an exclusive signaling molecule which functions downstream of IL-4 and IL-13 receptors. Thus, it is one of the attractive drug targets to suppress both IL-4 and IL-13 pathways, thereby reducing Th2 differentiation and suppressing the direct effects of these cytokines on airway tissues. This may lead to reduced airway hyperresponsiveness.
We have been utilizing STAT6-hormone binding domain of estrogen receptor fusion protein (STAT6-ER) to elucidate the role of STAT6. Estrogen analog 4-hydroxy tamoxifen (4-HT) can induce dimerization of the hormone binding domains, thereby resulting in the dimerization and activation of STAT6 without affecting other signaling pathways functioning in the downstream of IL-4 or IL-13 receptors. We could show that up-regulation of CD23 and MHC II in B cells can be induced by the activation of STAT6 alone. In addition, the differentiation of Th2 cells can be induced by the activation of STAT6 together with TCR signaling. We introduced STAT6-ER into airway epithelial cell lines and detected the induction of several chemokines upon STAT6 activation, suggesting a critical role of STAT6 in IL-4 and IL-13 receptor signaling in airway epithelium. To analyze the role of STAT6 in the intact animal, STAT6-ER transgenic mice are being produced.
Dominant negative STAT6 fused to the hormone binding domain of the estrogen receptor (dnSTAT6-ER) was also constructed. We could show that biological effects of STAT6 described above are suppressed in 4-HT dependent manner when dnSTAT6-ER was exogenously expressed. The suppressing effect of dnSTAT6-ER was not observed in other STAT family pathways, suggesting that the specificity is maintained in spite of the structural similarity of STAT family proteins. We are trying to produce transgenic mouse expressing dnSTAT6-ER in order to evaluate its suppressing activity on various biological responses involving STAT6, especially allergic responses and airway hyperresponsiveness.

3, The differential functions of NFAT family proteins in T cell activation

Jingtao Chen, Shoichiro Miyatake1 and Ken-ichi Arai1
1CREST(Japan Science and Technology)

NFAT family proteins have been shown to be required for the transcriptional activation of various cytokine genes in mature T cell lines, where three NFAT genes, NFATp, NFATc and NFATx are expressed. NFATp is the most abundant, while NFATc is upregulated by the activation through TCR and the expression level of NFATx is relatively low. Potent immunosuppressants, cyclosporin A (CsA) and FK506, exert their functions through inhibiting Ca-dependent phosphatase, calcineurin, thereby suppressing the activation of NFAT proteins. However, gene disruption experiments of each NFAT family member did not reveal absolute requirements of NFAT proteins for the transcriptional activation of various cytokine genes, suggesting that each NFAT protein may have overlapping functions. Furthermore, these experiments have suggested inhibitory functions of NFATp and NFATx in cytokine gene expression in T cells.
Deletion analysis of the NFAT homology domain (NHD) of NFATx lead to generation of a constitutively active mutant whose transcriptional activity is comparable to that of the activated wild type protein. To elucidate specific functions of NFATx in normal T cells, transgenic mice expressing this constitutively active NFATx in T lineage cells were produced. CD4 T cells isolated from the transgenic mice can express several cytokines including IL-2 and proliferate by PMA treatment alone. In addition, the cytokine production and proliferation are resistant to CsA treatment. These data strongly suggest that NFATx can stimulate transcription of several cytokine genes and cell proliferation. We are applying the same strategy to other NFAT family members to analyze their specific functions. NFATx is expressed at a high level in double positive population in thymus. The number of double positive thymocytes in NFATx -/- mice was reduced, indicating the requirement of NFATx for the survival of double positive thymocytes. Analysis of thymocytes from this transgenic mice expressing a ﾒgain-of-functionﾓ mutant will complement the data from a ﾓloss-of-functionﾓ mutant and may reveal additional roles of NFATx in thymic development.

II. Signaling mechanisms of cytokine receptors and during development

We have been analysing signal transduction mechanisms of cytokine receptors using various hematopoietic cells and fibroblasts. On the basis of accumulated knowledge on and techniques for cell signaling events, we have recently started to analyse molecular mechanisms of organogenesis. We use the zebrafish system in addition to mouse genetics with particular focus on eye development and hematopoietic cell differentiation.

1. Nuclear transport of Stat5

Zeng Rong, Yutaka Aoki, Saori Sato1, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology)

Stats play various critical roles in cytokine and growth factor signaling. In response to factor stimuli, Stats are phosphorylated and translocate to the nucleus, followed by activation of target genes. A portion of the Stats are degraded but most are dephosphorylated and relocate back to the cytoplasm where it can be reactivated. Using Ba/F3 cells expressing the hGM-CSF receptor, we found that nuclear export of Stat5B by factor-depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor CRM1. CRM1 binds to target proteins through the leucine-rich short stretch termed NES (nuclear export sequence). Mutation of putative NES of Stats resulted in disruption of translocation of Stat5B to cytoplasm after cytokine depletion. Interestingly, addition of LMB in the absence of cytokine lead to accumulation of Stat5B in nucleus in Ba/F3 and COS7 cells, suggesting that Stat5B shuttles between nucleus and cytoplasm in the CRM1-dependent manner regardless of cytokine stimulation. The mutant Stat5B lacking tyrosine 699, which is essential for Stat dimerization, behaves as the wild type, suggesting that shuttling between nucleus and cytoplasm in the absence of GM-CSF occurs in a monomer state. Using a series of mutant Stat5B, we identified a part of coiled coil (amino acids 138-165) as a critical region for monomer nuclear import. Interestingly, the N-terminal 104 amino acids are essential for dimer import of Stat5B. Taken together, we propose that two different import systems and the CRM1-dependent export system are coupled for regulation of Stat subcellular localization under various physiological states.

2. Cell cycle dependent interaction of Mad2 with conserved box1/2 region of hGM-CSF receptor common bc

Mitsuo Takeda1, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology)

Box1 is a conserved motif in the membrane proximal region of cytokine receptors, including the GM-CSF receptor. Studies using mutants of the hGM-CSF receptor bc as well as dominant negative Jak2 showed an essential role of Jak2 in hGM-CSF receptor signaling through its interaction with the box1 region of bc. Since overexpression of the wild type Jak2 inhibits hGM-CSF-induced proliferation, molecules other than Jak2 interacting with the box1 region may play roles in cell proliferation. Therefore, we searched for molecules interacting with the box1/2 region by pull-down assay using GST fused with the bc box1/2 region and the Ba/F3 cell lysate. Microsequence of a protein which associates with the box1/2 region but not with the hGM-CSFR a cytoplasmic region revealed that it is the mouse homologue of Mad2 protein which plays an important role in regulation of cell division. Peptides corresponding to the box1/2 region sequence also bind to Mad2 and a mutation of the conserved amino acid of box1 decreased Mad2 interaction. Deletion analysis of Mad2 indicated that interaction with box1/2 occurs through the C-terminal portion of Mad2. Association of Mad2 with other proteins is known to be regulated during cell cycle. Indeed, its binding affinity to box1/2 increased in the late M phase. These results suggest a possibility that GM-CSF regulates the M phase checkpoint through interaction with Mad2.

3. Cloning of factors involved in anti-apoptosis or proliferation using dimerization libraries.

MD Golam Mohi, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology)

We showed previously that Jak or Stat can be activated by induced dimerization using GyraseB/coumermycin or ER/tamoxifen dimerization system. It now appears that many signaling molecules or transcription factors are activated by dimerization. Therefore, we applied these inducible dimerization systems to isolate new genes involved in factor-dependent cell proliferation or anti-apoptosis. For this purpose, we constructed GyrB or ER fused cDNA library in retroviral vectors. These cDNA libraries were introduced into Ba/F3 cells and the clones were selected on the basis of their ability to support factor-independent proliferation or survival. The cDNA inserts were rescued from the isolated clones by PCR using primers designed from the retroviral LTR promoter regions. The cDNA inserts were then sequenced, and are now being analysed by transfection into Ba/F3 cells.

4. Analyses of the promoter regions of the human IL-3 receptor a chain (IL-3Ra) and GM-CSF receptor a chain (GM-CSFRa) genes

Eiji Akagawa, Ken-ichi Arai1 and Sumiko Watanabe
1CREST (Japan Science and Technology)

The receptors for IL-3 and GM-CSF consist of a and b subunits. Using transgenic mice expressing hGMR, we previously showed that features of biological activities of cytokines are determined primarily by expression pattern of their ligand binding unit, a subunit, rather than specificity of signal transduction pathways of each cytokine. We have analyzed mechanism of transcriptional regulation of the hIL-3R a and GM-CSFR a subunits. RT-PCR analysis showed different expression pattern of IL-3 and GM-CSF receptors in various cell lines. We determined sequences of the 1.5 kb genomic DNA spanning the promoter region of IL-3Ra gene. 5'-RACE and primer extension analyses indicated the presence of multiple transcription start sites. Using transient transfection assays, two regions (-363 ~ -331 and -124 ~ -76) of the hIL-3Ra promoter have been implicated in promoter enhancing activity. Electrophoresis mobility shift assays revealed specific binding of unknown protein(s) and Sp1 to these regions, suggesting roles of these proteins in hIL-3Ra gene induction. On the other hand, in the 2.5kb GM-CSF a promoter segment, the promoter-proximal PU-1 binding site was found to have transcriptional enhancing activity. These results suggested distinct mechanisms of transcriptional activation for expression of the IL-3 and GM-CSF receptors.

5. Analyses of transgenic mice expressing mutant hGM-CSF receptor

Eishun Muto, Yutaka Aoki, Koichiro Tsuji3, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology), 3Department of Clinical Oncology, Institute of Medical Science University of Tokyo

Using Tg mice expressing hGM-CSFR (wild-Tg), we previously showed that hGM-CSF is not a GM-lineage promoting factor but a strong proliferation promoting factor of all the lineages examined. GM-CSF induced not only GM colonies, but also the erythroid colonies were observed within bone marrow cells of wild-Tg mice. In contrast, thymocyte of wild-Tg mice ceased development at the pre-T cell stage by the addition of GM-CSF, and thus it is speculated that the GM-CSF may negatively regulate T cell development at a certain stage in wild-Tg mice. To analyse signaling requirement for these activities, we generated Tg mice expressing Fall hGM-CSF mutant receptor, in which all the cytoplasmic tyrosine residues were replaced by phenylalanine. Methyl cellulose assays of bone marrow cells from Fall-Tg, wild-Tg and their litter mate showed that Fall induced lower but still significant numbers of myeloid colonies. Interestingly, no erythroid colonies were observed in BM cells of Fall-mice. In vivo administration of hGM-CSF to wild-Tg mice resulted in disappearance of double positive cells in thymocyte, whereas no effects were seen with Fall-Tg mice thymocyte under the same condition. In vitro FTOC culture further confirmed these results. Taken together, our results suggest that signaling pathways stimulated through the tyrosine residues of hGM-CSF receptor may be required for erythroid colony formation and inhibition of thymocyte development.

6. Role of Stat3 in eye development

Akihiko Muto, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology)

Stat3 is a transcription factor known to be activated by various cytokines. We found that Stat3 protein was expressed in mouse lens and retina and the expression pattern in retina dramatically changed during development. In lens, Stat3 was mainly detected in epithelial cells in any stages. On the other hand, Stat3 was ubiquitously expressed in whole retinal cells at early developmental stage (E11), whereas in later stages (from E15 to P1) its expression becomes significant in the inner (ganglion) layer. In adult mice, Stat3 was exclusively detected in inner and outer plexiform layers, ganglion layer, nerve fiber and retinal inner segment. To analyze the role of Stat3 in eyes, we generated retina or lens-specific stat3-KO mice by using the Cre-loxP system. Morphology of the eyes and the expression of several retinal cell-type specific markers in the KO-mice were indistinguishable from those in control littermates. More detailed 　functional analyses are in progress.

7. Alteration of eye development by lens-specific expression of diphtheria toxin A

Ryo Kurita, Rika Ishida, Chiaki Abe, Hiroshi Sagara4, Ken-ichi Arai1, Sumiko Watanabe
1CREST (Japan Science and Technology), 4Department of Fine Morphology, Institute of Medical Science University of Tokyo

To understand the role of lens in development of other modules of eye, we designed a series of experiments in which to perturb lens development by expressing genes of interest under the control of the aA-crystallin promoter. We isolated the zebrafish counter part of aA-crystallin coding region by degenerate PCR and used this fragment to clone the 5ﾕ promoter region by screening genomic libraries. We fused the 2.5 kb 5ﾕ promoter region with EGFP (aAcrys-EGFP). Injection of the aAcrys-EGFP construct at the two cell stage revealed that this segment is sufficient for lens-specific expression of EGFP. Using this assay system, we mapped a minimal region responsible for lens-specific expression. This region contains a sequence homologous to that of 5ﾕ promoter region of the chicken aA-crystallin gene which includes a L-maf binding site. To perturb lens formation, we expressed diphtheria toxin A fragment under the regulation of the isolated aA-crystallin promoter. A plasmid containing aA-crystallin promoter-diphtheria toxin A was injected into eggs at the two cell stage. At 54 hours post fertilization, structural abnormalities of the lens was observed. Several vacuoles were observed within lens epithelial cells and an abnormal spaces were detected between the lens epithelium and fibers. Detailed phenotypes of the lens as well as other modules of the eye and the expression pattern of diphtheria toxin A are being investigated.

III. Studies on regulation of DNA replication

DNA replication is one of the central events of cell growth, and needs to be precisely regulated to ensure coordinated and ordered duplication of the entire genome within a specific period of the cell cycle, namely S phase. It is also under strict regulation of various external signals such as growth/differentiation factors and DNA damaging agents. Ordered assembly of protein complexes at replication origins is likely to be prerequisite for initiation of DNA replication in eukaryotes as well, as has been demonstrated for prokaryotic replicons. Recent studies in yeasts and frog eggs indicate cell cycle stage-specific assembly of replication-competent complexes during G1 phase. These complexes, termed prereplicative complexes (preRC), need to be ﾒtriggeredﾓ for DNA replication to be initiated. This triggering signal appears to be activated by cell cycle signals during G1 phase or by growth factor stimulation. The Cdc7-Dbf4 kinase and Cyclin-dependent kinase complexes are known to play essential roles to fire the origins.
It is well established that Cdks are conserved widely in eukaryotes and play critical roles at various stages of cell cycle. Saccharomyces cerevisiae Cdc7 kinase is essential for initiation and progression of S phase. Its kinase activity, which is controlled by the regulatory subunit Dbf4, peaks at the G1/S boundary. This oscillation of Cdc7 kinase activity is caused mainly by altered abundance of the Dbf4 protein which is regulated at both transcriptional and post-translational levels. We previously showed that Cdc7-related kinases and their regulatory subunits are conserved in higher eukaryotes including human, mouse and frog. This suggests that the regulatory mechanisms of initiation of DNA replication by Cdc7-related kinases may be conserved among all eukaryotes.
This year, we tried to elucidate how Cdc7 and Cdk may collaborate to achieve precise regulation of G1-S transition. We have obtained two possibly important results suggestive of collaboration and close interaction of these two essential kinases. First, phosphorylation of MCM2, the critical subunit of Cdc7, is facilitated by prior phosphorylation of critical serine residues on MCM2 by Cdk. Second, early embryonic lethality of mouse Cdc7 knockout can be partially rescued by elevating Cdk activity. These results indicate collaboration of the two kinases for initiation of S phase.
We are also interested in modes of DNA replication observed after ongoing replication forks are stalled. We already reported that E. coli PriA protein, a Zinc finger helicase, plays crucial roles in recombinational repair of double-stranded DNA breaks. PriA protein, widely conserved in eubacteria, specifically binds to structures resembling recombination intermediates or stalled replication forks. On the basis of detailed structure-function studies of PriA, we ultimately hope to find a protein with similar activity from eukaryotes in order to obtain clues on modes of replication that occur on damaged DNAs or at stalled replication forks.

1. Structure and function studies of ASK, the regulatory subunit for mammalian Cdc7 kinase.

Noriko Sato, Megumi Sato1, Ken-ichi Arai1 and Hisao Masai
1CREST (Japan Science and Technology)

Human Cdc7 (huCdc7)-ASK kinase complex, the human homologue of budding yeast Cdc7-Dbf4, is essential for chromosomal DNA replication in mammalian cells. ASK, whose expression fluctuates during cell cycle, binds and activates the huCdc7 catalytic subunit. In order to elucidate how huCdc7 kinase is regulated, we have generated a series of ASK mutants and analyzed their functions. We have identified three stretches of amino acids, namely Dbf4-motif-N, -M and C, which are conserved among the known Dbf4-related molecules. Small amino acid stretches containing either Dbf4-motif-M or Dbf4-motif-C can bind to huCdc7. For activation of huCdc7, the 177 amino acid region of ASK (minimum ASK) containing only motif-M and motif-C is sufficient. Mammalian ASK possesses a long C-terminal tail, which is not found in the yeast counterparts. This region can also bind to huCdc7 with high affinity and contains a cluster of autophosphorylation sites at its C-terminus. Cell cycle-specific phosphorylation of human MCM2 causes its characteristic mobility shift on SDS-PAGE during S to G2 phases. Overexpression of huCdc7 and ASK, either full-length or the minimum form, in COS7 cells can cause similar mobility shift of the endogenous MCM2. Furthermore, overexpression of minimum ASK in 293 cells enhances endogenous Cdc7 kinase activity. Currently, we are investigating the effect of overexpression of various mutant ASKs on DNA synthesis and cell cycle progression of mammalian cells.

2. Growth regulation of ASK, a regulatory subunit for human Cdc7-related kinase, is mediated by a 65 base-pair segment containing putative Sp1 sites and unknown repressive elements.

Masayuki Yamada, Noriko Sato, Kiyoshi Ohtani5, Ken-ichi Arai1 and Hisao Masai
1CREST (Japan Science and Technology), 5Human Gene Sciences Center, Tokyo Medical and Dental University

Transcription of ASK, a regulatory subunit for human Cdc7 kinase, is repressed in quiescent cells and is induced by growth stimulation during the cell cycle progression. In order to analyze modes of the transcriptional regulation of the ASK gene, we have isolated the promoter region of the ASK gene, and showed that it lacks a canonical TATA box but contains a cluster of E2F and Sp1 binding motifs near the transcription start sites. Using the luciferase reporter gene, we confirmed the induction of promoter activity in serum-stimulated NIH3T3 cells in transient transfection assays. Cotransfection of a plasmid expressing an E2F transcription factor into Ba/F3 cells, mouse IL-3 dependent proB cells, together with this reporter plasmid led to promoter activation without IL-3 stimulation. Furthermore, endogenous ASK mRNA was induced by infection of a recombinant adenovirus expressing E2F1. However, introduction of point mutations into putative E2F binding sites resulted in only slightly reduced response to serum stimulation and ectopically expressed E2F1, and did not result in constitutive activation, which is often observed with other E2F-regulated promoters. Promoter analyses using various deletion constructs have indicated that the most proximal Sp1 site plays a critical role in responses to E2F1 as well as to serum stimulation. These results suggest that transcription of the ASK gene during the transition from quiescence to proliferation is regulated by cooperation of E2F and Sp1. Furthermore, the 65 bp segment containing the essential Sp1 site and the major transcription initiation site, which is highly conserved between human and mouse, is sufficient for responses to serum stimulation and E2F.
The most striking property of E2F proteins is that they can drive quiescent cells into S phase. In order to examine whether deregulated expression of ASK protein can induce S phase in quiescent cells, we generated stable cell lines constitutively expressing ASK protein. The stable cell lines overexpressing ASK protein after factor depletion showed a population of S phase cells greater than that of the parental Ba/F3 cells under the same condition, suggesting that overexpression of ASK protein can partially bypass the mitogen requirement. Our data is consistent with the possibility that ASK is one of the critical targets of E2F for S phase induction.

3. Human Cdc7-related kinase complex: In vitro phosphorylation of MCM by concerted actions of Cdks.

Etsuko Matsui1, Yukio Ishimi6, Katsuyuki Tamai7, Ken-ichi Arai1, and Hisao Masai
1CREST (Japan Science and Technology), 6Mitsubishi Kasei Institute of Life Sciences, 7Moleclar Biology Laboratores, Inc.

We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione-S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and 4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM complex for initiation of S phase. Critical Cdk-mediated phosphorylation sites were mapped at serine 27 or serine 41 of mouse MCM2. Substitution of these serine residues by alanine resulted in very inefficient generation of the Cdc7-directed mobility-shifted form of MCM2 in vivo and in vitro, whereas that with glutamic acid lead to efficient phosphorylation of MCM2 by Cdc7. These results indicate that phosphorylation of MCM2 in MCM complex is achieved by concerted actions of Cdk and Cdc7 for efficient initiation of DNA replication.

4. Phosphorylation of MCM2 by Cdc7 kinase.

Min-kwon Cho, Etsuko Matsui1, Noriko Sato, Keiko Ogino1, Tadayuki Takeda1, Chika Taniyama1, Yukio Ishimi6, Ken-ichi Arai1, and Hisao Masai
1CREST (Japan Science and Technology Corporation); 6Mitsubishi Kasei Institute of Life Sciences

Genetic and biochemical evidence indicates that MCM2 is a critical substrate of Cdc7 kinase for firing of origins. Although both the free form of MCM2 and the one in a complex can be efficiently phosphorylated by Cdc7 in vitro, tryptic peptide mapping and mobility-shift on SDS-PAGE indicate phosphorylation of distinct residues on MCM2 in the complex. Analyses of MCM2 within the cells indicate that MCM2 in a complex is the major target of Cdc7 in vivo.
We have expressed and purified mouse MCM2-4-6-7 complexes containing a mutant MCM2 protein in which selected serine and threonine residues were replaced by alanine. We analyzed biochemical characteristics of these mutant MCM complexes and examined their phosphorylation by huCdc7-ASK kinase complex in vitro.
We have identified several in vitro phosphorylation sites on MCM2 using oligopeptides and recombinant proteins containing segments of MCM2 protein. Serine 17 of human MCM2 can be specifically phosphorylated by huCdc7 in vitro. A C-terminal region of the MCM box (F6), conserved in MCM family, and the region further C-terminal to it (NF3), which is unique to MCM2 but is conserved in MCM2 from other species, are also phosphorylated by huCdc7-ASK in vitro, which are confirmed by two-dimensional tryptic phosphopeptide mapping. Amino acid substitution of the corresponding serine/threonine residues in Cdc19 (fission yeast MCM2 homologue) with alanine or glutamic acid, respectively, resulted in loss of its function, as determined by the ability to complement the growth defect of cdc19(ts) mutant. Alanine substitutions of conserved serine/ threonine residues near the zinc-finger motif (F1) on full-length MCM2 resulted in loss of specific phosphorylation by huCdc7, and MCM2-4-6-7 complex containing the F1 mutation showed retarded migration of the complex on a native gel, indicating that F1 mutation causes alteration of complex structures.
Our results indicate that multiple residues on MCM2 are phosphorylated by Cdc7 and suggest a possibility that Cdc7 may regulate the subunit organization of the MCM complex.

5. Genetic studies on murine Cdc7-related kinase by the use of conditional knock-out ES cells and mice.

Jung Min Kim, Kazuki Nakao8, Kenji Nakamura8, Motoya Katsuki8, Nobuyuki Yamashita, Osamu Hatano9, Ken-ichi Arai1 and Hisao Masai
1CREST (Japan Science and Technology Corporation); 8DNA Biology and Embryo Engineering, Institute of Medical Science University of Tokyo; 9Nara Prefectural Medical School

In order to address in vivo functions of mammalian Cdc7 kinase, we generated muCdc7-deficient mice. muCdc7 homozygous null mice are early embryonic lethal (between E3.5 and 6.5). We have established muCdc7-/- ES cell lines and muCdc7-/- mice whose viability is maintained by the Cre-removable muCdc7 transgene encoding one of the cDNA variant (p62 driven by EF1a promoter). muCdc7-/- ES cells carrying the transgene showed growth properties indistinguishable from those of the wild-type ES cells. The transgene in this mutant ES cells was efficiently excised upon infection of Cre-encoding adenovirus, and the cell growth was immediately retarded with concomitant loss of thymidine incorporation into mutant cells, which eventually lost viability. The cells arrest mostly with S phase DNA content with concomitant increased expression of p53. These results demonstrate that muCdc7 is essential for proliferation and DNA replication of ES cells. In order to examine interactions between CDK and Cdc7 pathways in cell proliferation and mouse development, we tried to generate muCdc7-/- p27-/- double knockout mice. Viable embryos were detected at E8.5. Furthermore, muCdc7-/- p27-/- blastocysts formed inner cell mass which showed significant level of BrdU uptake, whereas muCdc7-/- p27+/+ blastocysts did not generate any visible inner cell mass nor DNA replication. These results indicate that increase of CDK activity can rescue early embryonic growth of muCdc7-/- embryos by partially restoring DNA replication activity in the absence of Cdc7 functions.
In contrast to the above mutant ES cells, the mutant mice with the identical genetic background were dead at one or two days after birth in about half the population, and those mice which survived were extremely small in size. The embryonic fibroblast cells derived from these mutant mice exhibited retarded growth in vitro, indicating that the transgene, although capable of rescuing the growth of muCdc7-/- ES cells, cannot completely rescue the loss of muCdc7 gene in more differentiated cells. We have identified many alternatively spliced forms of muCdc7 transcripts as well as the proteins derived from them, which are differentially expressed in various tissues. We are currently dissecting the roles of each variant in proliferation and functions of various tissues.
We are also developing more efficient systems for generating conditional knock-out ES cells and are trying to genetically dissect the functions of various cell cycle and DNA replication genes.

6. A novel mode of kinase activation through bipartite binding of a kinase activator to the catalytic subunit: Dissection of the regulatory subunit for Cdc7-related kinase essential for S phase.

Keiko Ogino1, Tadayuki Takeda1, Etsuko Matsui1, Hiromi Iiyama1, Chika Taniyama1, Ken-ichi Arai1 and Hisao Masai
1CREST (Japan Science and Technology Corporation)

Him1/Dfp1 protein of fission yeast, Schizosaccharomyces pombe, encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression of S phase. Him1 protein, containing three conserved motifs, Dbf4-motif-N, Dbf4-motif-M, and Dbf4-motif-C, binds and activates Hsk1 kinase which phosphorylates MCM2 protein. We have shown that Dbf4-motif-M and Dbf4-motif-C are essential for mitotic functions of Him1/Dfp1 protein as well as for full-level activation of the Hsk1 kinase. Deletion or mutation of either of Dbf4-motif-M or Dbf4-motif-C results in impaired mitotic function and kinase activation, although binding to Hsk1 is not affected. In vitro, a small segment containing Dbf4-motif-M (60 amino acids) alone or Dbf4-motif-C (58 amino acids) alone binds to Hsk1. Thus, Him1/Dfp1 binds to Hsk1 through bipartite binding modules. Furthermore, a fusion polypeptide containing small segments of Dbf4-motif-M and Dbf4-motif-C could activate Hsk1 and was capable of rescuing the growth defect of him1 null cells, and insertion of a long stretch of amino acids between the motif-M and motif-C could be tolerated for mitotic functions. In vitro, coexpression of two small segments of motif-M and motif-C could partially activate Hsk1. These results indicate a novel mode of kinase activation through bipartite binding of these conserved motifs to the catalytic subunit. Internal deletion of Dbf4-motif-N, which shares some similarity with the BRCT (BRCA C-terminal domain) motif, resulted in defect in hydroxyurea (HU)-induced checkpoint responses and sensitivity to methyl methane sulfonate (MMS), but mitotic functions and kinase activation were intact. Our results define the roles of the conserved motifs of Cdc7 regulatory subunits and suggest a molecular architecture of Cdc7-Dbf4 related kinase complexes at the origins.

7. A novel ﾒinitiation-typeﾓ mutant of fission yeast Cdc45 homologue reveals its essential role in loading of DNA polymerase a onto MCM.

Masashi Uchiyama, Dominic Griffiths10, Ken-ichi Arai1 and Hisao Masai
1CREST (Japan Science and Technology Corporation), 10ICRF, United Kingdom

Proteins involved in the initiation of DNA replication play critical roles in the loading and assembly of replication complexes at replication origins. To gain novel insights into the regulation of initiation, we screened for fission yeast temperature sensitive mutants which arrest at G1/S boundary. One of the isolated mutants, goa1-U53, arrests after START but before the hydroxyurea block point. This mutant is allelic to the CDC45 homologue of fission yeast (sna41+) and mutated in a motif highly conserved in Cdc45-related proteins. goa1-U53 genetically interacts with DNA polymerase a temperature sensitive mutants. The gene product of the cloned cDNA complementing goa1-U53 (Sna41p/SpCdc45p) interacts with Polap throughout cell cycle and with Mis5p/Mcm6p in the chromatin fraction during S phase. Although Polap loading onto the chromatin fraction, which occurs before START, is not affected, association of Polap with Mis5p is absent in goa1-U53. These results support a model that loading of DNA polymerase a onto replication origins occurs through two distinct steps, namely START-independent loading onto the chromatin at early G1 and association with the replication complexes at the origins at G1/S boundary through S phase in a manner dependent on Cdc45 which interacts with both DNA polymerase a and MCM.

8. PriA protein in recombination-dependent DNA replication from stalled replication forks.

Taku Tanaka, Chika Taniyama1, Ken-ichi Arai1, and Hisao Masai
1CREST (Japan Science and Technology Corporation)

E. coli PriA protein, a DEXH-type DNA helicase with unique C2C2-type zinc finger-like motifs interrupting the helicase domains, is an essential component of the fX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. We have postulated that PriA protein recognizes stalled replication forks to restore replication forks and repair legions in a recombination-dependent manner. We show here that mutant PriA proteins defective in ATP hydrolysis and helicase activities do not fully support iSDR or cSDR in vivo, although the ATPase/helicase-deficient mutants could support single-stranded DNA replication from the origin of fX174, as was reported previously. We have also identified a novel motif in the N-terminal segment of PriA, WYY motif, which, along with a C-terminal helicase conserved motif, is required for binding to a D-loop structure, mimicking the stalled replication forks. This motif is also required for iSDR and cSDR in vivo. Thus, ATPase/ helicase and D-loop binding activities are both required for recombination-dependent DNA replication. We are currently fractionating fission yeast and mice tissue extracts to identify eukaryotic proteins which recognize the stalled replication forks.

9. Mapping of replication origins in human and mouse cytokine cluster regions.

Kanako Osawa, Vesna Todorovic11, Lisako Tsuruta, Naofumi Takemoto1, Yumiko Kamogawa1, Naoko Arai2, Ken-ichi Arai1, Mauro Giacca11, Shoichiro Miyatake and Hisao Masai
1CREST (Japan Science and Technology Corporation), 2Department of Immunology, DNAX Research Institute, 11International Centre of Genetic Engineering and Biotechnology, Trieste, Italy

The mode of activation of mammalian replication origins is still poorly understood. In spite of various evidence pointing to interplay between replication and transcription, the molecular basis for coordination between these two nucleic acid transactions is still largely an open question. The human chromosome 5 and mouse chromosome 11 contain the genome regions in which cytokine genes are present in clusters. We are particularly focusing on the human 5q locus containing tandemly arranged IL-3 and GM-CSF genes and mouse 11q containing IL-4 and IL-13 genes as models for the study of possible coupling between transcription and replication. Expression of IL-3 and GM-CSF in T cells is induced in response to T cell receptor stimulation. IL-4 and IL-13 belong to type 2 T helper cell-specific cytokines, and their expression is strictly cell-type specific. By utilizing an origin-enriched library as well as the competitive PCR method, we have identified a replication origin which is active in non-T cell lines at 4 kb downstream of human GM-CSF gene. We did similar mapping in T cell lines, and preliminary results indicate the presence of an origin in the region between the two genes, upstream of GM-CSF. We are currently mapping replication origins in the vicinity of the mouse IL-4 and IL-13 locus in Th2 cells, Th1 cells and non-T cells. We wish to extend these studies to eventually determine genome-wide distribution pattern of replication origins in various cell types.

Pan, S., Tsuruta, L., Masuda, E.S., Imamura, R., Bazan, F., Arai, K., Arai, N. and Miyatake, S. (2000) ﾒNFATz: A novel Rel similarity domain containing proteinﾓ Biochem. Biophys. Res. Comm. 272, 765-776

Lee, H.J., Takemoto, N., Kurata, H., Kamogawa, Y., Miyatake, S., OﾕGarra, A. and Arai, N. (2000) ﾒGATA-3 induces T helper cell type2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cellsﾓ J. Exp. Med. 192, 105-115

Miyatake, S., Arai, N. and Arai, K. (2000) ﾒChromatin remodeling and T helper subset differentiationﾓ IUBMB Life 49, 473-478

Takemoto, N., Kamogawa, Y., Lee, H.J., Kurata, H., Arai, K., OﾕGarra, A. and Miyatake, S. (2000) ﾒCutting Edge: Chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene clusterﾓ J. Immunol. 165, 6687-6691

Ryo, K., Kamogawa, Y., IIkeda, I., Yamauchi, K., Yonehara, S., Nagata, S. and Hayashi, N. ﾒSignificance of Fas Antigen-mediated apoptosis in human fluminant hepatic failure. ﾓ American journal of gastroenterology, (2000), 95, 2047-2055

Harada, Y., Miyatake, S., Arai, K., Watanabe, S., (2000) Cyclic AMP inhibits the activity of c-Jun N-terminal kinase 2 (JNK2) but not JNK1 and ERK2, in human helper T lymphocytes. Biochem. Biophys. Res. Comm. 266, 129-134

Liu, R., Liu, C.-B., Mohi, M.G., Arai, K., Watanabe, S. (2000) Signals for prevention of g irradiation-induced apoptosis by hGM-CSF. Oncogene, 19, 571-579

Dahl, M. E., Arai, K., Watanabe, S. (2000) Association of Lyn tyrosine kinase to the GM-CSF and IL-3 receptor common bc subunit and role of Src tyrosie kinases in DNA synthesis and anti-apoptosis., Genes to Cells, 5, 143-153

Liu, R., Arai, K., Watanabe, S. (2000) Antiapoptotic activity of the hGM-CSF receptor. J. Allergy Clin. Immunol., 106, 10-18

Watanabe, S., Aoki, Y., Nishijima, I., Xu, M.-J., Arai, K. (2000) Analysis of signals and functions of the chimeric hGM-CSF receptor in BA/F3 cells and transgenic mice. J. Immunol., 164, 3653-3644

Sato, T., Maekawa, T., Watanabe, S., Tsuji, K., Nakahata, T., (2000) Erythroid Progenitors Differentiate and Mature in Response to Endogenous Erythropoietin, J. Clinical Invest. 106, 263-270.

Okubo, T., Yanai, N., Watanabe, S., Arai K.-I. and Obinata M. (2000) Effect of human granulocyte-macrophage colony stimulating factor (hGM-CSF) on lymphoid and myeloid differentiation of sorted hematopoietic stem cells from hGM-CSF receptor gene transgenic mice. J. Biochem., 127, 591-596

Sakurai, Y., Arai, K., Watanabe, S. (2000) In vitro analysis of mechanism of STAT5 activation by granulocyte-macrophage colony-stimulating factor. Genes to Cells. 5, 937-947

Watanabe, S., Rong Zeng, Yutaka Aoki, Tohru Itoh, Ken-ichi Arai, (2001) Initiation of polyoma virus origin dependent DNA replication through STAT5 activation by human granulocyte-macrophage colony-stimulating factor (GM-CSF). Blood in press.

Masai, H., and Arai, K. Dbf4 motifs: conserved motifs in activation subunits for Cdc7 kinases essential for S-phase. Biochem. Biophys. Res. Commun., 18, 228-232. 2000

Masai, H., Matsui, E., You, Z., Ishimi,Y., Tamai, K. and Arai, K. Purification and characterization of human Cdc7-ASK kinase complex expressed in insect cells: In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7 by Cdks.　J. Biol. Chem., 275, 29042-29052, 2000

Masai, H. and Arai, K. Regulation of DNA replication during cell cycle: Roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest. IUBMB Life, 49, 353-364, 2000

Johnstone, L., Masai, H. and Sugino, A. A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans. Prog. Cell Cycle Res. 4, 61-69, 2000

Takeda, T., Ogino, K., Tatebayashi, K. Ikeda, H., Arai, K. and Masai, H. Regulation of initiation of DNA replication and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast.　Mol. Biol. Cell, in press

Masai, H., Kim, J-M., and Arai, K. Cdc7 kinase complex: A key regulator for initiation of DNA replication.　Journal of Cellular Physiology, in press

Masai, H., Tanaka, T. and Arai, K. PriA: a DNA helicase which recognizes recombination intermediates in responses to replication fork blocks.　Protein and Peptide Science, in press

Masai, H. Bacterial primosome, Encyclopedia of Life Sciences (Macmilan Reference Limited), 2001, in press

Masai, H. Bacterial replication fork: synthesis of lagging strand, Encyclopedia of Life Sciences (Macmilan Reference Limited), 2001, in press

サイトカインシグナル伝達機構、鴨川由美子、新井賢一、"喘息" 2000、Vol.13 No4, p89-97

武藤彰彦、渡辺すみ子CD116, CD123, 臨床免疫34、466−471，2000

伊藤暢、渡辺すみ子　CD131、臨床免疫　34、550−555，2000

正井久雄、内山雅司、Jung Min Kim、竹田忠行、熊谷啓之、佐藤憲子、新井賢一：染色体複製の開始を制御する因子、実験医学　増刊　「細胞周期研究のフロンティア」（中山敬一、正井久雄、佐方功幸　編集）羊土社、pp127-136, 2000