Construction of an open-access database that integrates cross-reference information from the transcriptome and proteome of immune cells.

MOTIVATION: Although a huge amount of mammalian genomic data does become publicly available, there are still hurdles for biologists to overcome before such data can be fully exploited. One of the challenges for gaining biological insight from genomic data has been the inability to cross-reference transcriptomic and proteomic data using a single informational platform. To address this, we constructed an open-access database that enabled us to cross-reference transcriptomic and proteomic data obtained from immune cells. RESULTS: The database, named RefDIC (Reference genomics Database of Immune Cells), currently contains: (i) quantitative mRNA profiles for human and mouse immune cells/tissues obtained using Affymetrix GeneChip technology; (ii) quantitative protein profiles for mouse immune cells obtained using two-dimensional gel electrophoresis (2-DE) followed by image analysis and mass spectrometry and (iii) various visualization tools to cross-reference the mRNA and protein profiles of immune cells. RefDIC is the first open-access database for immunogenomics and serves as an important information-sharing platform, enabling a focused genomic approach in immunology. AVAILABILITY: All raw data and information can be accessed from http://refdic.rcai.riken.jp/. The microarray data is also available at http://cibex.nig.ac.jp/ under CIBEX accession no. CBX19, and http://www.ebi.ac.uk/pride/ under PRIDE accession numbers 2354-2378 and 2414.

Mammalian polyhomeotic homologues Phc2 and Phc1 act in synergy to mediate polycomb repression of Hox genes.

The Polycomb group (PcG) gene products form multimeric protein complexes and contribute to anterior-posterior (A-P) specification via the transcriptional regulation of Hox cluster genes. The Drosophila polyhomeotic genes and their mammalian orthologues, Phc1, Phc2, and Phc3, encode nuclear proteins that are constituents of evolutionarily conserved protein complexes designated class II PcG complexes. In this study, we describe the generation and phenotypes of Phc2-deficient mice. We show posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts associated with derepression of Hox cluster genes and Cdkn2a genes, respectively. Synergistic actions of a Phc2 mutation with Phc1 and Rnf110 mutations during A-P specification, coimmunoprecipitation of their products from embryonic extracts, and chromatin immunoprecipitation by anti-Phc2 monoclonal antibodies suggest that Hox repression by Phc2 is mediated through the class II PcG complexes, probably via direct binding to the Hox locus. The genetic interactions further reveal the functional overlap between Phc2 and Phc1 and a strict dose-dependent requirement during A-P specification and embryonic survival. Functional redundancy between Phc2 and Phc1 leads us to hypothesize that the overall level of polyhomeotic orthologues in nuclei is a parameter that is critical in enabling the class II PcG complexes to exert their molecular functions.

Absence of DNA polymerase theta results in decreased somatic hypermutation frequency and altered mutation patterns in Ig genes.

Multiple DNA polymerases participate in somatic hypermutation of immunoglobulin (Ig) genes. Mutations at A/T are largely dependent on DNA polymerase eta (POLH) whereas mutations at C/G appear to be generated by several DNA polymerases. We have previously shown that mice expressing a catalytically inactive POLQ (Polq-inactive) have a reduction in C/G mutations. Here we have generated mice that completely lack Polq expression (Polq-null). Polq-null mice have no obvious abnormality in B or T cell differentiation, and their splenic B cells responded normally to various activation signals and underwent normal Ig gene class switching. The mutant mice mounted relatively normal immune responses against a T-dependent antigen although there was a slight decrease in antigen specific antibodies. Polq-null mice exhibited a mild reduction in the overall mutation frequency, however, in contrast to Polq-inactive mice where the reduction mostly affected mutations at C/G, Polq-null mice showed a reduction of both C/G and A/T mutations and there was a significant increase of G to C transversions. These results confirm a role for POLQ in somatic hypermutation and suggest that in the complete absence of POLQ other polymerases may functionally substitute, resulting in a mutation pattern different from that found in Polq-inactive mice.

Up-regulation of the error-prone DNA polymerase {kappa} promotes pleiotropic genetic alterations and tumorigenesis.

It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberrations, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase kappa, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase kappa favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype.

Role of Clast1 in development of cerebellar granule cells.

We have identified the murine Clast1/LR8 gene by subtraction of cDNA derived from CD40 ligand-activated and naive B cells. The Clast1 gene is ubiquitously expressed in various organs of adult mice. However, its physiological function was largely unknown. To study a role of Clast1, we established Clast1-deficient (Clast1-KO) mice. Here, we reveal that approximately 65% of Clast1-KO mice showed severe ataxia. The Clast1-KO cerebellum with ataxia is small in size and revealed a severely aberrant lobulation, loss of the internal granule cell layer, and the disorganized Purkinje cells. Clast1 mRNA is expressed in the cerebellar granule cells of normal adult mice. Developmentally, Clast1 mRNA is also detected in the external germinal layer of the embryonic cerebellum, indicating its expression in granule cell precursors. Histopathological analysis of the developing Clast1-KO cerebellum demonstrated the reduced number of cells in the external germinal layer. Thus, Clast1 is required for development of cerebellar granule cells.

FcRY, an Fc receptor related gene differentially expressed during B lymphocyte development and activation.

A bioinformatics approach has lead to the identification of FcRY, a new Fc receptor related gene. FcRY is predicted to encode a protein with three immunoglobulin (Ig) domains followed by a mucin-like domain containing a proline-rich stalk and a C-terminal leucine rich region. The predicted protein lacks a hydrophobic domain for insertion into the plasma membrane, suggesting that FcRY is an intracellular or secreted protein. This feature is shared with the product of the FcRX/FCRL/FREB gene that is closely linked to FcRY in both human and mouse genomes. Fcry transcripts are first detectable among mouse B lineage cells at the pre-B cell stage. Splenic B cells of the newly formed, follicular, and marginal zone subsets express Fcry, as do germinal center B cells to a lesser extent. FcRY is also expressed in subpopulations of human B cells. A consistent characteristic of FcRY in both species is low level gene expression, which can be further downregulated in normal mouse B cells by signaling through the B cell receptor (BCR) or CD40, thereby suggesting a correlation between cell cycle entrance and diminished FcRY expression. Fcry is upregulated by short-term treatment with BAFF/BLyS, which promotes B cell survival rather than proliferation. LPS induces very rapid but transient enhancement. We observed a pronounced upregulation of Fcry expression in WEHI 231 cells induced by BCR crosslinking to undergo cell cycle arrest prior to apoptosis, consistent with the possible regulation of Fcry expression by cell cycle status.

Fas ligand-expressing tumors induce tumor-specific protective immunity in the inoculated hosts but vaccination with the apoptotic tumors suppresses antitumor immunity.

The interaction between Fas and Fas ligand (FasL) is involved in the apoptotic death of a number of cells including lymphocytes. Forced expression of FasL in tumors can induce apoptosis of infiltrating Fas-positive T cells; accordingly, tumors can survive in the milieu of systemic immune responses. However, FasL-expressing murine lung carcinoma (A11) and melanoma (B16) cells did not develop subcutaneous tumors and FasL-expressing A11 (A11/FasL) cells produced few spontaneous lung metastatic foci in syngeneic mice. The mice that rejected A11/FasL cells were resistant to subsequent challenge of parent A11 but not irrelevant B16 cells. Vaccination of mice with UV-treated A11/FasL, but not UV-treated A11 cells, however, augmented the growth rate of A11 but not B16 tumors, both of which were subsequently inoculated. The number of lung metastatic foci of A11 cells was also increased in the mice that received UV-treated A11/FasL but not UV-treated A11 cells. Intraperitoneal injection of UV-treated A11/FasL cells resulted in the production of larger amounts of immunosuppressive TGF-beta in peritoneal exudate than that of UV-treated A11 cells. Expression of the CD80 costimulatory molecule in tissues where UV-treated A11/FasL cells were inoculated was lower than the expression at an untreated A11/FasL-injected site. Our results indicated that apoptotic FasL-expressing tumor cells could impair host immune responses against the tumors, in contrast to potent antitumor immunity generated by viable FasL-expressing tumors.

T-cell-dependent antitumor effects produced by CD40 ligand expressed on mouse lung carcinoma cells are linked with the maturation of dendritic cells and secretion of a variety of cytokines.

CD40/CD40 ligand (CD40L) interaction plays an essential role in cell-mediated immune responses. We examined whether expression of CD40L in murine lung carcinoma (A11) cells could produce antitumor effects. The proliferation rate in vitro of A11 cells transfected with the murine CD40L gene (A11/CD40L) was not different from that of parent cells; however, half of the immunocompetent mice inoculated with A11/CD40L cells did not form tumors and the growth of A11/CD40L tumors developed in the rest of mice was significantly retarded compared with that of parent tumors. Protective immunity was also induced in the mice that had rejected A11/CD40L cells. In T-cell-defective nude mice, these antitumor effects were not observed. Bone-marrow-derived dendritic cells (DCs), when cultured with A11/CD40L cells, formed clusters with the tumors and showed upregulated CD86 expression. Expression of the interleukin-23 (IL-23) p19, IL-12p35, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with A11/CD40L but not with A11 cells, and P40, the subunit of both IL-12 and IL-23, was secreted from the cocultured DCs. These data directly showed that the expression of CD40L in tumors facilitated the interaction between DCs and the tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity.

Stage-specific expression of Clast6/E3/LAPTM5 during B cell differentiation: elevated expression in human B lymphomas.

We have identified a CD40-regulated gene, Clast6/E3/LAPTM5, by subtraction of cDNAs derived from resting and CD40 ligand-treated B cells. Clast6/E3/LAPTM5 is abundantly expressed in resting mature B cells but is rapidly and transiently repressed by treatment with CD40 ligand, a T helper signal that induces B cell activation. Using a fluorescence activated cell sorter, we have purified B-lineage cells into distinct populations based on their differential expression of cell surface markers. Clast6/E3/LAPTM5 was found to be highly expressed in progenitor and precursor B cells, downregulated in late pre-B and immature B cells, and upregulated again in mature B and the germinal center B cells. Interestingly, Clast6/E3/LAPTM5 was expressed at high levels in malignant B lymphomas. These results reveal stage-specific expression of Clast6/E3/LAPTM5 during B cell differentiation and implicates its possible involvement in B cell malignancies.

Elevated expression of DNA polymerase kappa in human lung cancer is associated with p53 inactivation: Negative regulation of POLK promoter activity by p53.

DNA polymerase kappa (POLkappa) is a low fidelity translesional DNA polymerase implicated in spontaneous and DNA damage-induced mutagenesis. We have previously shown that POLkappa was frequently overexpressed in human lung cancer tissues as compared with their matched non-tumorous tissue counterpart. In the present study, we found a close correlation between elevated POLkappa expression and p53 inactivation in lung cancer tissues. To investigate whether POLK expression might be regulated by p53, we have determined the transcriptional initiation site of POLK gene and examined its promoter activity in A549, H358-129, and PC-3 human lung cancer cell lines. Wild-type p53, but not a mutant p53 (R273H) devoid of the DNA-binding activity, strongly inhibited POLK promoter activity in these cells. In addition, POLK promoter exhibited a significantly higher activity in p53-/- murine embryo fibroblasts (MEF) than in p53+/- and p53+/+ MEF. These results link p53 status with POLkappa expression and suggest that loss of p53 function may in part contribute to the observed POLkappa upregulation in human lung cancers.

Expression of the TNF-alpha gene on mouse lung carcinoma cells suppresses spontaneous lung metastasis without affecting tumorigenicity.

Forced expression of TNF-alpha in tumor cells has been shown to inhibit their tumor growth in vivo through a number of mechanism such as activation of an immune system and induction of an apoptotic process. We re-examined the anti-tumor effects caused by the TNF-alpha gene transfer using high-metastatic, murine lung carcinoma A11 cells. Expressed TNF-alpha molecules remained on cell surface and were not secreted into culture supernatants in vitro. Syngeneic immunocompetent mice developed tumors of TNF-alpha-expressed A11 cells and the growth of their subcutaneous tumors was not different from that of parent tumors. Spleen of the mice that developed TNF-alpha-expressed A11 tumors was significantly larger than that of the mice bearing parent tumors, but relative ratios of each cell population were not different. In contrast to subcutaneous tumors, the number of spontaneous lung foci metastasized from the subcutaneous TNF-alpha-expressed A11 tumors was markedly reduced compared with that from parent tumors. Expressed TNF-alpha on tumors is released by matrix metalloproteinases from surrounding tissues and anti-tumor effects by TNF-alpha can be influenced by local environmental conditions.

Cell growth- and P53-dependent transcriptional activity of the midkine promoter confers suicide gene expression in tumor cells.

Midkine (MK) is preferentially expressed in a number of human tumors, while the expression in adult normal tissues is restricted. Previous studies showed that a 2.3-kb regulatory region of the human MK gene could selectively activate a linked suicide gene in tumors. In this study, we explored the minimal promoter region using genomic fragments deleted from the 5'-upstream side and analyzed the mechanism of the preferential activation in tumor cells. Luciferase assays showed that the 0.3-kb fragment from the transcription start site contained a cis-acting element(s) for the promoter activity. Expression of the herpes simplex virus-thymidine kinase gene under the control of the MK promoter followed by ganciclovir administration produced antitumor effects in vivo. Transfection of the wild-type p53 gene into the immortalized fibroblasts bearing mutated p53 and tumor cell lines, which induced cell cycle arrest, decreased the MK promoter-mediated transcription more effectively than the SV40 or the cytomegalovirus promoter-mediated transcription. The P53-mediated downregulation of the MK promoter activity was stronger in p53-defective tumors than in wild-type p53-bearing tumors. Moreover, the MK promoter-mediated luciferase activity was greater in p53-deficient mouse embryonic fibroblasts than in those bearing wild-type p53 gene. The transcriptional activity of the MK promoter could be regulated by cell growth and in part P53-dependent pathways.

T cell dependent and independent antitumor immunity generated by the expression of Fas ligand on mouse lung carcinoma cells.

Interaction between Fas and Fas ligand (FasL) induces apoptotic cell death of Fas-positive cells. Expression of FasL on tumors therefore possibly kills activated Fas-positive cytotoxic T cells that infiltrated into the tumors and consequently the tumors can evade from systemic immune responses. Previous studies however showed that forced expression of FasL in tumors induced neutrophil-mediated inflammatory reactions and accordingly produced T cell independent antitumor effects in the inoculated animals. We then analyzed the FasL-mediated antitumor responses with genetically mutated mice. Murine lung carcinoma (A11) cells transfected with the FasL gene (A11/FasL), which was able to kill Fas-positive B cells, did not form subcutaneous tumors and produced few lung spontaneous metastatic foci in immunocompetent mice. The mice that rejected A11/FasL cells developed tumor-specific protective immunity. A11/FasL cells were also rejected in T cell-defective nude mice and in CD18-deficient mice which showed impaired neutrophil functions, but not in Fas-defective (lpr/lpr) mutant mice. Antitumor activities on A11 cells were dependent on the number of co-injected A11/FasL cells but those on irrelevant B16 murine melanoma cells were not produced even with a large number of co-injected A11/FasL cells. In contrast to previous reports, the present study implies that T cells can also be effectors of FasL-mediated antitumor responses and neutrophils are not absolutely required for the responses.

Antitumor effects are produced by forced expression of membrane-bound but not soluble Fas ligand in murine lung carcinoma cells.

Interaction of Fas and Fas ligand (FasL) in immunocompetent cells plays a crucial role(s) in their effector functions and in the regulation of host immune responses. Expression of FasL in tumors possibly counteracts Fas-positive effector T cells that infiltrate into tumors and consequently the Fas/FasL interaction can contribute to the escape of tumor cells from systemic immune systems. However, forced expression of FasL in tumors unexpectedly induced migration of neutrophils into the tumors and the FasL-expressing tumors were rejected due to the inflammatory reaction. Since FasL is released from the cell surface, we examined whether soluble or membrane-bound FdsL molecules produced such antitumor effects. Fas-positive B cells were effectively killed by membrane-bound but not soluble FasL in which the leader sequence of interleukin-4 was ligated with the extracytoplasmic portion of FasL. Mice inoculated with A11 murine lung cancer cells expressing membrane-bound FasL did not develop tumors and had few spontaneous lung metastatic foci. In contrast, mice injected with A11 cells secreting soluble FasL developed tumors; the growth of the tumors and the number of lung metastatic foci from subcutaneous tumors were not different from those of parent tumors. The chemotactic activity of FasL, tested by intraperitoneal injection of parent and the FasL-expressing A11 cells, showed that the level of neutrophil migration by A11 cells secreting soluble FasL was greater than that by parent cells but was not as significant as that by A11 cells expressing membrane-bound FasL. The antitumor activity induced by expressed FasL seems to be correlated with the apoptosis-inducing activity through the Fas/FasL interaction but not directly with the chemotactic activity for neutrophils.

DNA polymerases eta and theta function in the same genetic pathway to generate mutations at A/T during somatic hypermutation of Ig genes.

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

Role of DNA polymerase theta in tolerance of endogenous and exogenous DNA damage in mouse B cells.

DNA polymerase theta (Poltheta) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Poltheta in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Poltheta polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, gamma-irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Poltheta is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Poltheta in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes.

DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes.

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase eta (Poleta) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J(H)4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase.

A role for c-fos/activator protein 1 in B lymphocyte terminal differentiation.

Expression of B lymphocyte-induced maturation protein 1 (Blimp-1) transcription factor is essential for promoting B cell differentiation into plasma cells. However, a critical transcription factor for Blimp-1 expression in activated B cells is unclear. When splenic B cells were stimulated with CD40 ligand (CD40L) and IL-4, terminal differentiation was induced in the B cells from c-fos transgenic (H2-c-fos) mice but barely in those from control littermates and from c-fos-deficient mice. AP-1 family and Blimp-1 mRNAs were transiently induced in the control B cells, and overexpression of c-Fos induced a sufficient amount of Blimp-1 for terminal differentiation in the H2-c-fos B cells. When normal and c-fos-deficient B cells were stimulated with LPS, a sufficient amount of Blimp-1 for terminal differentiation was induced in those B cells. However, expression of c-fos/AP-1 family mRNAs in LPS-stimulated normal B cells was similar to that of normal B cells stimulated with CD40L and IL-4. EMSA and chromatin immunoprecipitation assays using the AP-1-binding DNA sequence in the murine Blimp-1 promoter region demonstrated that AP-1-binding activity in nuclear protein of LPS-stimulated normal B cells was prolonged more than that in normal B cells stimulated with CD40L and IL-4. Furthermore, the percentage of CD138(+) B cells within germinal center B cells in the spleen and the number of Ab-forming cells in the bone marrow of H2-c-fos mice was larger than that of control mice 12 days after immunization. Thus, although c-Fos is not essential for Blimp-1 expression, c-Fos/AP-1 positively regulates Blimp-1 expression and terminal differentiation of activated B cells.

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death.

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.

Clast5/Stra13 is a negative regulator of B lymphocyte activation.

CD40 is a member of the tumor necrosis factor receptor family and mediates a variety of functions of B cells, including B cell survival, proliferation, immunoglobulin gene class switching, memory B cell formation, and regulation of Fas-mediated apoptosis. To begin to elucidate the molecular mechanism governing such diverse functions of CD40, we have isolated a gene from mouse splenic B cells, termed Clast5, whose expression is strongly repressed during B cell activation. Clast5 is identical with Stra13, a recently identified member of the basic helix-loop-helix family of transcription factors. Clast5/Stra13 is highly expressed in unstimulated, resting B cells and is rapidly downregulated by a variety of stimuli that activate B cells, including CD40 ligand, anti-IgM antibodies, lipopolysaccharides and interleukin-4. Forced expression of Clast5/Stra13 in B cells delayed the cell cycle progression into S phase and strongly suppressed Fas-mediated apoptosis. Moreover, Clast5/Stra13 inhibited the colony formation in fibroblasts. Our results suggest that Clast5/Stra13 functions as a negative regulator of B cell activation by inhibiting cell cycle progression and cell growth.