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The Runx1 transcription factor cooperates with or antagonizes other transcription factors and plays essential roles in the differentiation and function of T lymphocytes. Previous works showed that Runx1 is expressed in peripheral CD4(+) T cells which level declines after T cell receptor (TCR) activation, and artificial deletion of Runx1 causes autoimmune lung disease in mice. The present study addresses the mechanisms by which Runx1 contributes to the maintenance of peripheral CD4(+) T cell quiescence. Microarray and quantitative RT-PCR analyses were employed to compare the transcriptome of Runx1 -/- CD4(+) T cells to those of unstimulated and TCR-stimulated Runx1 +/- cells. The results identified genes whose expression was modulated similarly by Runx1 deletion and TCR activation. Among them, genes encoding cytokines, chemokines, and Jak/STAT signaling molecules were substantially induced. In Runx1-deleted T cells, simultaneous increases in Il-17A and Rorγc, a known master gene in TH17 differentiation, were observed. In addition, we observed that the loss of Runx1 reduced the transcription of genes encoding quiescence-associated transcription factors, including Foxp1, Foxo1, and Klf2. Interestingly, we identified consensus Runx1 binding sites at the promoter regions of Foxp1, Foxo1, and Klf2 genes, which can be enriched by chromatin immunoprecipitation assay with an anti-Runx1 antibody. Therefore, we suggest that Runx1 may activate, directly or indirectly, the expression of quiescence-associated molecules and thereby contribute to the maintenance of quiescence in CD4(+) T cells.

Runx1 transcription factor is a key player in the development and function of T cells. Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naive T cells from C57BL/6 mice, but its expression was remarkably induced upon T-cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from −412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T-cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signaling-induced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin–NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation.

The RUNX family genes encode transcription factors that are involved in development and human diseases. RUNX1 is one of the most frequently mutated genes in human hematological malignancies and is a critical factor for the generation and maintenance of hematopoietic stem cells. Another Runx family gene, Runx3, is known to be expressed in hematopoietic cells. However, its involvement in hematopoiesis remains unclear. Here we show the hematopoietic phenotypes in Runx3 conditional knockout (KO) mice (Runx3(fl/fl);Mx1-Cre(+)): whereas young Runx3 KO mice did not exhibit any significant hematopoietic defects, aged Runx3 KO mice developed a myeloproliferative disorder characterized by myeloid-dominant leukocytosis, splenomegaly, and an increase of hematopoietic stem/progenitor cells (HSPCs). Notably, Runx3-deficient cells showed hypersensitivity to granulocyte-colony stimulating factor, suggesting enhanced proliferative and mobilization capability of Runx3-deficient HSPCs when stimulated. These results suggest that, besides Runx1, Runx3 also plays a role in hematopoiesis.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G1 cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G1-phase extension and represses CD8 expression in a G1 extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G1 extension and CD8 repression. Importantly, forced G1 extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G1 progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G1-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

Mast cells (MCs) are developed from hematopoietic progenitor cells and play an important role in inflammation. Study of the kinetics of development and accumulation of primate MC in vivo is crucial for the control of human inflammatory diseases, as evolution of the immune system is quite rapid and inflammation including MC response is considered to be different between mouse and human. In the present study, we examined the development of MC from hematopoietic progenitors of Callithrix jacchus (common marmoset), an experimental animal of nonhuman primates. Bone marrow cells were fractionated for the expression of CD34 and CD117 by cell sorting. MCs were developed in vitro or by transplanting the cells to NOD/SCID/IL-2γc knockout (NOG) mice. In vitro culture of CD34(+)CD117(+) (double positive, DP) cells with stem cell factor could generate high-affinity Fc epsilon receptor (FcεR)-expressing CD117(+) cells with typical granules. The developed MC released β-hexosaminidase and produced leukotriene C(4) after the stimulation of FcεRI. Transplantation of DP cells gave rise to a marked expansion of CD34(-)CD45(+)CD117(+)FcεR(+) cells in NOG mice. They expressed transcripts encoding chymase 1 and tryptase β. Differentiation of CD34(-)CD117(+) cells to MCs was relatively limited compared with the DP cells, similarly to human MCs. These results suggest that this marmoset system provides a good model for human MC development.

Cells in the immune system are regulated positively or negatively by sets of receptor pairs that conduct balanced, activating, or inhibitory intracellular signaling. One such receptor pair termed paired Ig-like receptor (PIR) is composed of the inhibitory PIR-B and its activating isoform, PIR-A. Upon binding to their shared ligand, MHC class I molecules, these receptors control the threshold for immune cell activation. Gene-targeting studies on PIR-B in mice revealed the importance of the inhibition mediated by the PIR-B–MHC interaction in the immune system. Recent studies also revealed the significance of the interaction of PIR-B with neurite outgrowth inhibitors, including Nogo in the CNS. The coordinated regulation by PIR-B and PIR-A is considered to be primarily dependent on their expression balance in cells. However, the mechanism underlying transcriptional control of the genes for PIR-B and PIR-A (Pirb and Pira, respectively) remains to be clarified. In this study, we identified the major cis-acting promoter segment for Pirb and Pira in B cells as the −212 to −117 region upstream from the translation initiation codon. PU.1 and Runx3 were found to bind to this Pirb promoter. Truncation of the PU.1-binding motif significantly reduced the promoter activity, whereas the influence of elimination of the Runx3 site was marginal in B lymphoma BCL1-B20 cells. Unexpectedly, PU.1, but not Runx3, knockdown reduced the levels of both the Pirb and Pira transcripts. We conclude that the major promoter of Pirb, and probably Pira as well, is activated dominantly by PU.1 and marginally by Runx3 in B cells.

Over the past years, increasing numbers of distinct subsets have been discovered and identified for a T lymphocytes' entity. Differentiation and function of each T cell subset are controlled by a specific master transcription factor. Importantly, Runt-related transcription factors, particularly Runx1 and Runx3, interplay with these master regulators in various aspects of T cells' immunity. In this review article, we first explain roles of Th-Pok and Runx3 in differentiation of CD4 versus CD8 single positive cells, and later focus on cross-regulation of Th-Pok and Runx3 and their relationship with other factors such as TCR strength. Next, we provide evidences for the direct interplay of Runx1/3 with T-bet and GATA3 during Th1 versus Th2 commitment to activate or silence transcription of signature cytokine genes, IFNγ and IL4. Lastly, we explain feed-forward relationship between Runx1 and Foxp3 and discuss roles of Runx1 in regulatory T cells' suppressive activity. This review highlights an essential importance of Runx molecules in controlling various T cell subsets' differentiation and functions through molecular interplay with the master transcription factors in terms of protein-protein interaction as well as regulation of gene expression.

OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated.We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure.Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation.The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3.OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.

Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

Runx family proteins play indispensable roles in the development of various hematopoietic lineage cells. However, their function in NK cells is still uncertain. We found that NK cells and CD8 T cells dominantly express Runx3 protein, whereas NKT cells and CD4 T cells express Runx1. Reverse transcription-PCR analysis revealed that Runx3 expression is initiated at the NK precursor stage and is maintained along the course of NK cell differentiation. In order to examine their role in the earlier stage of NK cell development, we introduced Runx dominant-negative (Runx dn) form into Lin(-)c-kit(+)Sca-1(+) hematopoietic stem cells, which were applied to NK cell-inducing culture. Post-cultured cells showed a decreased expression of IL-2/IL-15 common receptor beta subunit (CD122), consistent with another finding that Runx binds to promoter region of CD122 gene. To examine the Runx function in the later developmental stage, we used transgenic mouse, in which Runx dn form is expressed in immature and mature NK cells. This mouse showed decreased expressions of NK maturation markers, such as Ly49 family, Mac-1 and CD43, whereas IFN-gamma production was greatly enhanced. These findings suggest that Runx proteins, especially Runx3, play multiple roles in NK cell differentiation.

The death or survival of double positive (DP) thymocytes is determined by the strength of their TCR signaling. Of the three Runx family proteins, the DP cells only express the Runx1 transcription factor. We introduced and expressed in murine thymocytes the Runt domain of Runx1, which antagonizes the activity of endogenous Runx1. The Runt transgenic DP thymocytes expressed higher levels of the proapoptotic molecules Fas and Bim compared with the wild-type cells. Furthermore, the Runt transgenic cells were more susceptible to apoptosis induced by the artificial cross-linking of the TCR by the anti-CD3 Ab. This susceptibility was partially abrogated by the lpr/lpr background. In addition, Runx1:HY-TCR-double transgenic DP thymocytes were resistant to the apoptosis induced by the endogenously presented HY Ag. We propose that Runx1 functions to suppress the apoptotic sensitivity of DP thymocytes in the context of TCR signaling.

To illuminate the origins of NADPH oxidase (Nox), we identified cDNA clones encoding Nox2, Nox4, p22 phagocyte oxidase (phox), p47phox, and p67phox in a chordate phylogenetically distant to the vertebrates, the sea squirt Ciona intestinalis. We also examined the spatiotemporal expression of these genes in embryos and juveniles. The sequences of the Nox2, Nox4, p22phox, p47phox, and p67phox cDNAs contained open reading frames encoding 581, 811, 175, 461, and 515 amino acids, respectively. The level of identities between the deduced Nox2, Nox4, p22phox, p47phox, and p67phox amino acid sequences and their corresponding human components were 54.0, 31.0, 44.4, 36.0, and 26.2%, respectively. Despite these low identities, the functional domains of the C. intestinalis and human NADPH oxidase and Nox4 are highly conserved. The genomic organizations of the components of the NADPH oxidase gene except for p67phox (a single exon gene) and the Nox4 gene in C. intestinalis are highly similar to those of the corresponding human NADPH oxidase genes. Further, the analyzed part of the C. intestinalis genome and EST database do not seem to present p40phox and Nox5. The Nox2, p22phox, p47phox, and p67phox genes were specifically expressed in the blood cells of juveniles. The Nox4 gene was expressed in blood cells and endostyle of juveniles. These results suggest that C. intestinalis NADPH oxidase components possess potential functional activities similar to those of human, but the manner in which cytosolic phox proteins in C. intestinalis interact is different from that in human.

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.

T cell development is dependent on the integration of multiple signaling pathways, although few links between signaling cascades and downstream nuclear factors that play a role in thymocyte differentiation have been identified. We show here that expression of the HMG box protein TOX is sufficient to induce changes in coreceptor gene expression associated with β-selection, including CD8 gene demethylation. TOX expression is also sufficient to initiate positive selection to the CD8 lineage in the absence of MHC–TCR interactions. TOX-mediated positive selection is associated with up-regulation of Runx3, implicating CD4 silencing in the process. Interestingly, a strong T cell receptor–mediated signal can modify this cell fate. We further demonstrate that up-regulation of TOX in double positive thymocytes is calcineurin dependent, linking this critical signaling pathway to nuclear changes during positive selection.

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12p11, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.

The immunity provided by ascidian hemocytes represents one prototype of innate immune function in vertebrates. However, there are currently no molecular markers of ascidian hemocytes. We accumulated a large number of ESTs of cDNAs derived from hemocytes of Ciona intestinalis, a cosmopolitan species of ascidian. By comparing these ESTs with those derived from other tissues and developmental stages of Ciona, we were able to extract 81 transcripts expressed abundantly and preferentially in hemocytes. Among them, the von Willebrand factor type A (vWA)-like and complement 6 (C6)-like transcripts were found to be expressed almost exclusively in hemocytes, based on RT-PCR analysis and whole mount in situ hybridization. We propose that vWA-like and C6-like can be used as molecular markers for Ciona hemocytes.

During thymic T cell development, immature CD4+/CD8+ thymocytes develop into either CD4+/CD8− helper or CD4−/CD8+ CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8α strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4−/CD8+ T cells. Instead, mature cells with a CD4+/CD8+ phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.

Ascidians, which are classified as urochordata, appear to employ a primitive system of host defense that is considered to be a prototype of vertebrate innate immunity. We performed a cDNA/EST study to identify the genes expressed in the hemocytes of Ciona intestinalis. We obtained 3357 one-path reads that were then grouped into 1889 independent clusters. Although two thirds of the clusters could not be assigned to any particular gene, the remaining 530 clusters had significant homology to genes with known function. Of these, 62 clusters appeared to be related to host defense mechanisms. These include transcripts whose products are probably involved in cytotoxicity, detoxification, inflammation, and apoptosis. As expected, elements of acquired immunity were not detected. Thus, Ciona hemocytes appear to express a number of host defense-related genes involved in innate immune mechanisms.

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CD8 SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CD8 SP cells.

Recent studies revealing that endothelial cells derived from E8.5-E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known. We found that mice deficient in Runx1/AML1 (an α subunit of the transcription factor PEBP2/CBF) lack c-Kit+ haematopoietic cell clusters in the dorsal aorta, omphalomesenteric and umbilical arteries, as well as yolk sac vessels. Moreover, endothelial cells sorted from the embryo proper and the yolk sac of AML1–/– embryos are unable to differentiate into haematopoietic cells on OP9 stromal cells, whereas colonies of AML1–/– endothelial cells can be formed in culture. These results strongly suggest that the emergence of haematopoietic cells from endothelial cells represents a major pathway of definitive haematopoiesis and is an event that also occurs in the yolk sac in vivo, as suggested by earlier in vitro experiments.

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.

Mice lacking the AML1/PEBP2alphaB/CBFa2 gene or PEBP2beta/CBFb gene exhibit a defect in definitive hematopoiesis and die in utero because of hemorrhage in the central nervous system. Hematopoiesis in the embryo is considered to be tightly associated with vascular development. Here we examined whether PEBP2/CBF plays any role in angiogenesis besides that in definitive hematopoiesis. We found that AML1/PEBP2alphaB/CBFa2, PEBP2alphaA/CBFa1, and PEBP2beta/CBFb were expressed in a murine endothelial cell line MSS31. The expression of these molecules as well as the DNA binding activity of PEBP2/CBF were augmented by angiogenic growth factors such as bFGF and VEGF. Moreover, the expression of PEBP2 alpha/CBFa protein in endothelial cells was confirmed at the site of angiogenesis in vivo. To further clarify the role of PEBP2/CBF in angiogenesis, we established permanent transfectants of PEBP2 beta-MYH11 gene, one that interacts with the runt domain of the alpha subunit and deregulates PEBP2/CBF in a dominant interfering manner. Proliferation, migration, and tube formation of the PEBP2 beta-MYH11 transfectants were significantly reduced in comparison with those activities of the mock transfectants. These results suggest that transcription factor PEBP2/CBF plays an important role in angiogenesis.

The AML1 gene, which encodes the DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, is involved in several types of chromosomal translocations associated with human acute myeloid leukemia, and has been shown by gene targeting to be essential for the development of definitive hematopoiesis in the murine fetal liver. In addition, the gene is expressed abundantly in T lymphocytes and has been implicated in T cell specific gene expression. In the present study we examined the function of AML1 in T cell receptor (TCR)-mediated, Fas/Fas-ligand dependent apoptosis of a T hybridoma line, DO11.10. Several independent cell clones overexpressing the AML1 protein were isolated by transfecting AML1 cDNA into these cells. These clones possessed an increased level of PEBP2/CBF DNA binding activity and were found to be resistant to apoptosis induced by anti-CD3 antibody treatment. Northern blot analysis revealed that induction of the Fas-ligand transcript was markedly suppressed in the anti-CD3 treated clones. Instead, expression of IL-2 receptor alpha subunit (IL-2R alpha), which is a manifestation of proliferative TCR signaling, was induced. This was in contrast to the parental, anti-CD3 treated DO11.10 cells where induction of Fas-ligand but not of IL-2R alpha was observed. Resistance of the AML1 overexpressing cell clones to TCR-mediated apoptosis is most likely attributable to the lack of Fas-ligand induction, since simultaneous treatment with anti-CD3 and anti-Fas antibodies caused apoptosis of the clones. The overall results suggest that the AML1 protein may play a pivotal role in switching TCR signaling between apoptosis and cell proliferation in T lymphocytes.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM- CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13- acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.

The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1−/−Bcl2tg mice harboring Runx1-deleted CD4+ T cells developed a fatal autoimmune lung disease. CD4+ T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4+ lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4+ T cells and, thereby, maintains cell quiescence.

The mouse TCRγ locus is positively regulated by the transcription factors STAT5 and Runx. While the locus undergoes frequent rearrangements in T lymphocytes, TCRγ transcription is repressed in αβ T cells. This phenomenon, known as TCRγ silencing, depends on pre-TCR-induced thymocyte proliferation. The molecular basis for TCRγ silencing, however, is largely unknown. Here, we show that pre-TCR signaling reduces transcription and histone acetylation of the TCRγ locus irrespective of V-J rearrangements. We also demonstrate that Runx is recruited to Eγ and HsA enhancer elements of the TCRγ locus, primarily at the CD4(-)CD8(-) double-negative stage and that Runx binding to these elements decreases at later stages of thymocyte development. Importantly, anti-CD3 antibody treatment decreased IL-7R expression levels, STAT5 phosphorylation and recruitment of STAT5 and Runx to Eγ and HsA elements in RAG2-deficient thymocytes, suggesting that pre-TCR signaling triggers reduced binding of STAT5 and Runx to the enhancer elements. Furthermore, we observed that misexpression of STAT5 or Runx in the CD4(+)CD8(+) double-positive cell line DPK induces TCRγ gene transcription. Finally, we showed that TCRγ transcription is induced in αβ T cells from Runx3 transgenic mice, suggesting that Runx3 counteracts TCRγ silencing in αβ T cells in vivo. Our results suggest that pre-TCR signaling indirectly inactivates TCRγ enhancers by reducing recruitment of STAT5 and Runx and imply that this effect is an important step for TCRγ silencing in αβ T cells.

The IL-7R controls local accessibility of joining (J) gamma gene segments in the mouse TCRgamma locus by recruiting signal transducers and activators of transcription (STAT) 5 and transcriptional coactivators to the Jgamma germ line promoters and inducing histone acetylation and germ line transcription. Because STAT consensus motifs are conserved not only in the Jgamma promoters but also in the TCRgamma 3' enhancer (Egamma) elements, it is possible that STAT5 interacts with and activates Egamma. To address this question, we first showed that the lysine 4 residue of histone H3 is substantially methylated at Egamma1 and Egamma4 elements in wild-type early thymocytes and that the levels of the methylation are reduced in IL-7R alpha chain-deficient mice. We also showed that STAT5 has potential to elevate histone acetylation of the Egamma elements in a cytokine-dependent cell line by cytokine stimulation. Next, we demonstrated that STAT5 is recruited to the STAT consensus motifs in the Egamma elements after cytokine stimulation and that transcription factors Runt-related (Runx) and c-Myb are constitutively recruited to Egamma. Furthermore, we showed that STAT5 augments basal Egamma activity controlled by Runx and c-Myb. These results suggest that STAT5 is recruited to the consensus motifs in the Egamma elements by cytokine stimulation and augments basal Egamma activity independent of Runx and c-Myb. Therefore, this study implies that the Egamma elements might be activated in two successive steps, first by Runx and c-Myb and next by STAT5.

Element I, homologous to the adenovirus type 5 E1A enhancer core, is a 10-bp sequence in the A core of the polyomavirus enhancer and was shown previously to be responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that element I by itself was capable of activating polyomavirus DNA replication in COP-5 cells which express the polyomavirus large T antigen. A nuclear factor, polyomavirus enhancer-binding protein 5 (PEBP5), which bound to the entire sequence of element I and was responsive to TPA was identified by an in vitro binding assay. Although the binding site of PEBP5 partly overlaps with that of PEBP1 (PEA1), a member of the AP-1 family, PEBP5 appears to be a distinct factor. Since we previously showed that element I alone was able to activate transcription, our present results suggest that PEBP5 is involved in the regulation of both transcription and replication of DNA. The amount of PEBP5 increased after F9 cells were induced to differentiate by retinoic acid. A relatively large amount of PEBP5 was detected in lymphoid and trophoblast cells.

We have previously identified a protein factor, PEBP2 (polyomavirus enhancer-binding protein), in the nuclear extract from mouse NIH 3T3 cells which binds to the sequence motif, PEA2, located within the polyomavirus enhancer A element. Upon cellular transformation with activated oncogene c-Ha-ras, this factor frequently undergoes drastic molecular modifications into an altered form having a considerably reduced molecular size. In this study, the altered form, PEBP3, was purified to near homogeneity. The purified PEBP3 comprised two sets of families of polypeptides, alpha-1 to alpha-4 and beta-1 to beta-2, which were 30 to 35 kilodaltons and 20 to 25 kilodaltons in size, respectively. Both kinds of polypeptides possessed DNA-binding activities with exactly the same sequence specificity. Individual alpha or beta polypeptides complexed with DNA showed faster gel mobilities than did PEBP3. However, the original gel retardation pattern was restored when alpha and beta polypeptides were mixed together in any arbitrary pair. These observation along with the results of UV- and chemical-cross-linking studies led us to conclude that PEBP3 is a heterodimer of alpha and beta subunits, potentially having a divalent DNA-binding activity. Furthermore, PEBP3 was found to bind a second, hitherto-unnoticed site of the polyomavirus enhancer that is located within the B element and coincides with the sequence previously known as the simian virus 40 enhancer core homology. From comparison of this and the original binding sites, the consensus sequence for PEBP3 was defined to be PuACCPuCA. These findings provided new insights into the biological significance of PEBP3 and PEBP2.

Although the transcription factor AML1/Runx1 is known to be essential for definitive hematopoiesis, its role in T cell differentiation is not well understood. In this study, we investigated the functions of AML1 in the early stage of thymocyte differentiation. For this, we crossed AML1 dominant interfering form (Runt)-transgenic mice with TCR-transgenic mice, and demonstrated the decrease of CD4 + 8 + (DP) thymocyte cell number although their proportion was not reduced. Reaggregation culture system for thymocytes of (Runt×TCR) double transgenic mice, in which the rate of de novo transition from DN cells to the DP stage can be estimated, showed that the cell division during the DN-to-DP transition is impaired without significant cell death. These results indicate that AML1 is involved in thymocyte differentiation by controlling cell proliferation.

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.

We generated cell hybrids between mouse T cell lymphoma EL4 cells and mouse fibroblast B82 cells (BELIII and BELIV) to examine the expression of T cell receptor (TcR)/CD3 genes and their lymphoid-specific transcription factor genes, which are normally detected in EL4 cells. In BELIII and BELIV, expression of the TcR alpha, TcR beta and CD3 delta genes was extinguished, whereas expression of the CD3 epsilon gene was still detected. Expression of the (lymphoid enhancer binding factor 1) LEF-1 gene was extinguished and that of the GATA-3 gene was hardly detected in BELIII and BELIV. Ets-1 gene expression, observed not only in EL4 cells but also in B82 cells, was considerably reduced in BELIII and BELIV. A much higher level of PEBP2 alpha A gene expression was observed in B82 cells than in EL4 cells and was preserved in BELIII and BELIV. To examine whether reduced expression of these genes is also found in T cell x B cell hybrids, we generated an additional cell hybrid between EL4 cells and mouse plasmacytoma S194 cells (SELIII). Marked differences were observed in the expression of the TcR alpha, CD3 delta, LEF-1 and PEBP2 alpha A genes in BEL and SEL hybrids. Expression of the TcR alpha, CD3 delta and LEF-1 genes, which was extinguished in BELIII and BELIV, was detected in SELIII. PEBP2 alpha A gene expression, not detected in S194 cells, was considerably reduced in SELIII. Almost the sum of the chromosomes from the parental cells were retained by, and the presence of every gene was proven, in each cell hybrid. These results suggest that suppression of the expression of lymphoid-specific transcription factor genes may precede that of the TcR/CD3 genes in the cell hybrids, and that the presence of a different trans-acting negative regulatory mechanism(s) suppresses the expression of T cell specific genes in fibroblasts and B cells.

Definitive hematopoiesis begins in the para-aortic, splanchnopleural (P-Sp) and aorta-gonad-mesonephros (AGM) regions of mouse embryos and then switches to the fetal liver [1] [2] [3]. Gene-targeted mice lacking the c-Myb transcription factor have severe hematopoietic defects in the fetal liver [4]. The role of c-Myb, if any, in P-Sp/AGM hematopoiesis has not been examined, however. Recently, we reported that oncostatin M can effectively expand both hematopoietic and endothelial-like cells from in vitro cultures of the AGM region [5]. Using this cell culture system, we examined the involvement of c-Myb in definitive hematopoiesis in the P-Sp and AGM regions. When primary cultures from the P-Sp or AGM regions of wild-type mouse embryos were probed with an anti-c-Myb antibody, hematopoietic cells but not endothelial-like cells showed positive staining. In contrast, in the P-Sp/AGM culture from c-myb(-/-) embryos, no hematopoietic cells were generated and endothelial-like cells predominated, indicating that the impairment of hematopoiesis in the liver of c-myb(-/-) embryos is actually preceded by a defect in P-Sp/AGM hematopoiesis. Hematogenic precursor cells were, however, still present in an inert but competent form among the endothelial-like, adherent cell population of c-myb(-/-) P-Sp/AGM cultures. When infected with a retrovirus carrying c-myb cDNA, these cultures gave rise to a significant number of hematopoietic cells. The rescued cells, unlike wild-type hematopoietic cells, were negative for c-Kit (a marker of hematopoietic progenitors), but did express other hematopoietic cell surface markers such as Mac-1, Gr-1 (myeloid markers), CD19, B220, Thy-1.2 (Iymphoid markers), and Ter119 (an erythroid marker). Thus, c-Myb plays a role in the generation of hematopoietic cells in the embryonic P-Sp and AGM regions.

The function of the A element (nucleotides 5107 to 5130) of the polyomavirus enhancer is augumented in NIH 3T3 cells by a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). One of its targets is an AP1 consensus sequence motif recognized by a nuclear factor, PEBP1. In Ha-ras-transformed NIH 3T3 cells, however, A element function was not enhanced by TPA treatment, and at the same time PEBP1 was not detected in the nuclear extract by a mobility shift assay. PEBP1 was not detected in either the extract from NIH 3T3 cells treated in vivo with a protein kinase inhibitor, staurosporine, or the extract from NIH 3T3 cells after treatment in vitro with phosphatase. These results suggest that PEBP1 is required to be properly phosphorylated for DNA binding and that it is underphosphorylated, possibly due to the downregulation of protein kinase C in Ha-ras-transformed cells. In addition, we observed that PEBP2, which bound to the A element adjacent to PEBP1, was converted to apparently related PEBP3 when conditions favored underphosphorylation.

SummaryTo understand how CD8 expression is regulated during the transition process from CD4+8+ (CD4 and CD8 double positive, DP) to CD4−8+ (CD8 single positive, CD8SP) cells in the thymus, the involvement of Runx proteins in the alteration of chromatin configuration was investigated. Using the chromatin immunoprecipitation assay, we first demonstrated that Runx proteins bind to the stage-specific CD8 enhancer, as well as the CD4 silencer, in CD8SP thymocytes. Among Runx family members, Runx3 expression was initiated in DP thymocytes receiving a positive selection signal and increased in concert with differentiation to the CD8SP stage. Furthermore, reactivation of the CD8 gene, as well as CD4 silencing, was suppressed in positively selected thymocytes of Runx dominant-negative transgenic mice. These results suggest that Runx proteins, especially Runx3, are involved in lineage specification of CD8 T cells and provide important information for understanding the mechanism for the mutually exclusive expression of coreceptors in mature thymocytes.

A mutant of polyomavirus, F441, selected to grow in undifferentiated mouse F9 embryonal carcinoma cells, carries a single-base change in the enhancer region at nucleotide (nt) 5233 of the viral genome. Enhancers of most of the F9 mutants have a duplicated segment of viral DNA encompassing nt 5233. The minimum duplicated segment of all the known F9 mutants is from nt 5218 to nt 5239. We prepared oligonucleotides spanning the sequence from nt 5218 through nt 5239 of the genome of the wild type and F441 and examined the biological activities of the oligonucleotides by a transient assay of chloramphenicol acetyltransferase (CAT) gene expression in F9 cells. The oligonucleotide harboring the F441 mutation was shown to increase cat gene expression in F9 cells when linked at an upstream position in both orientations. When dimerized at an upstream position, the F441 oligonucleotide showed even higher cat gene expression enhancing activity. In contrast, no such effects were observed with the oligonucleotide of the wild-type sequence. In addition, the F441 oligonucleotide, but not the wild-type sequence, could inhibit the activity of whole enhancer fragment of F441 when cotransfected into F9 cells in excess amounts. On the basis of the results obtained, we suggest that the segment of F441 enhancer encompassing the point mutation contains a target for a cellular factor(s) which acts in a positive manner to increase the transcription of a gene in undifferentiated mouse F9 cells.

An mRNA sequence of two human respiratory syncytial viral nonstructural protein genes and of a gene for a 22,000-molecular-weight (22K) protein was obtained by cDNA cloning and DNA sequencing. Sequences corresponding to the 5' ends of the respective transcripts were deduced directly by primer extension and dideoxy nucleotide sequencing of the mRNAs. The availability of a bicistronic clone (pRSC6) confirmed the gene order for this portion of the genome. Contrary to other unsegmented negative-stranded RNA viruses, a 19-nucleotide intercistronic sequence was present between the NS1 and NS2 genes. The translation of cloned viral sequences in the bicistronic and monocistronic clones (pRSNS1 and pRSNS2) revealed two moderately hydrophobic proteins of 15,568 and 14,703 daltons. Their similarity in molecular size explained our earlier inability to resolve these proteins. A DNA sequence of an additional recombinant plasmid (pRSA2) revealed a long open reading frame encoding a 22,156-dalton protein containing 194 amino acids. It was relatively basic and moderately hydrophobic. A protein of this size was readily translated in vitro from a viral mRNA hybrid selected by this plasmid and corresponded to an unglycosylated 22K protein seen in purified extracellular virus but not associated with detergent- and salt-resistant cores. A second open reading frame of 90 amino acids partially overlapping with the C terminus of the 22K protein was also present within this sequence. This was reminiscent of the viral matrix protein gene which was previously shown by us to contain two overlapping reading frames. The finding of three additional viral transcripts encoding at least three identifiable proteins in human respiratory syncytial virus was a novel departure from the usual genetic organization of paramyxoviruses. The 5' ends of all three transcripts had a 5'NGGGCAAAU sequence that is common to all viral transcripts analyzed so far. Although there was no obvious homology immediately upstream of the polyadenylate tail, an AGUUA (AGUAA in the case of NS2) was present between 1 and 4 nucleotides upstream of the polyadenylate end of NS1 and 22K protein mRNAs.

A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly stimulates the activity of polyomavirus enhancer in a human erythroleukemia cell line, K562. The target of stimulation was the previously defined A element (from nucleotides 5107 to 5130) of the enhancer. We found that within the A element, two partly overlapping sequence motifs (one from nucleotides 5107 to 5117, the other from nucleotides 5113 to 5121) were independently the targets of TPA stimulation. The former is homologous to the enhancer core sequence of the adenovirus type 5 E1A gene, and the latter shares the consensus AP-1-binding site. In addition, transiently expressed Ha-ras oncogene also stimulated these two subelements in K562 cells, as we reported for NIH 3T3 cells previously.

The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4 − 8 + subset of T lymphocytes. In the thymus of Runx3 -deficient mice, CD4 expression is de-repressed and CD4 − 8 + thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4 − 8 + thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3 -transgenic thymus, the number of CD4 − 8 + cells was greatly increased, whereas the numbers of CD4 + 8 + and CD4 + 8 − cells were reduced. The CD4 − 8 + transgenic thymocytes contained mature cells with a TCR high HSA low phenotype. These cells were released from the thymus and contributed to the elevated level of CD4 − 8 + cells relative to CD4 + 8 − cells in the spleen. Runx3 overexpression also increased the number of mature CD4 − 8 + thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4 + 8 − lineage selection. Thus, Runx3 can drive thymocytes to select the CD4 − 8 + lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.