DNA is frequently damaged by genotoxic stresses such as ionizing radiation, reactive oxygen species, and nitrogen species. DNA damage is a key contributor to cancer initiation and progression, and thus the precise and timely repair of these harmful lesions is required. Recent studies revealed transcription as a source of genome instability, and transcription-coupled DNA damage has been a focus in cancer research. Impaired mRNA export is closely related to DNA damage through R-loop formation. The molecular machineries of transcription-coupled DNA damage have been extensively analyzed in Saccharomyces cerevisiae. However, the molecular basis of these phenomena in higher eukaryotes remains elusive. In this review, we focus on the relationship between deregulated mRNA export through the transcription-export-2 (TREX-2) complex and cancer development. Particularly, the expression of germinal center-associated nuclear protein (GANP), a molecular scaffold in the TREX-2 complex, is highly associated with tumorigenesis in mice and humans. Although the deregulated expression of other components in the TREX-2 complex might affect cancer development, we have directly demonstrated the significance of GANP in tumorigenesis using genetically modified mice. Additionally, we describe recent evidence for medical applications demonstrating that the downregulation of the other components may be a good candidate for a chemotherapeutic target in terms of reducing the side effects.

Perturbation of genes is important for somatic hypermutation to increase antibody affinity during B-cell immunity; however, it may also promote carcinogenesis. Previous studies have revealed that transcription is an important process that can induce DNA damage and genomic instability. Transciption-export-2 (TREX-2) complex, which regulates messenger RNA (mRNA) nuclear export, has been studied in the budding yeast Saccharomyces cerevisiae; however, recent studies have started investigating the molecular function of the mammalian TREX-2 complex. The central molecule in the TREX-2 complex, that is, germinal center-associated nuclear protein (GANP), is closely associated with antibody affinity maturation as well as cancer etiology. In this review, we focus on carcinogenesis, lymphomagenesis, and teratomagenesis caused by transcription-coupled DNA damage through GANP and other components of the TREX-2 complex. We review the basic machinery of mRNA nuclear export and transcription-coupled DNA damage. We then briefly describe the immunological relationship between GANP and the affinity maturation of antibodies. Finally, we illustrate that the aberrant expression of the components of the TREX-2 complex, especially GANP, is associated with the etiology of various solid tumors, lymphomas, and testicular teratoma. These components serve as reliable predictors of cancer prognosis and response to chemotherapy.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis.

A vaccine that can effectively prevent HIV-1 transmission remains paramount to ending the HIV pandemic, but to do so, will likely need to induce broadly neutralizing antibody (bnAb) responses. A major technical hurdle toward achieving this goal has been a shortage of animal models with the ability to systematically pinpoint roadblocks to bnAb induction and to rank vaccine strategies based on their ability to stimulate bnAb development. Over the past 6 years, immunoglobulin (Ig) knock-in (KI) technology has been leveraged to express bnAbs in mice, an approach that has enabled elucidation of various B-cell tolerance mechanisms limiting bnAb production and evaluation of strategies to circumvent such processes. From these studies, in conjunction with the wealth of information recently obtained regarding the evolutionary pathways and paratopes/epitopes of multiple bnAbs, it has become clear that the very features of bnAbs desired for their function will be problematic to elicit by traditional vaccine paradigms, necessitating more iterative testing of new vaccine concepts. To meet this need, novel bnAb KI models have now been engineered to express either inferred prerearranged V(D)J exons (or unrearranged germline V, D, or J segments that can be assembled into functional rearranged V(D)J exons) encoding predecessors of mature bnAbs. One encouraging approach that has materialized from studies using such newer models is sequential administration of immunogens designed to bind progressively more mature bnAb predecessors. In this review, insights into the regulation and induction of bnAbs based on the use of KI models will be discussed, as will new Ig KI approaches for higher-throughput production and/or altering expression of bnAbs in vivo, so as to further enable vaccine-guided bnAb induction studies.

Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells.

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10(3) pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.

Nuclear export of mRNPs is mediated by transport factors such as NXF1 that bind mRNPs and mediate their translocation through the central channel of nuclear pores (NPC) using transient interactions with FG-nucleoporins. A number of nuclear factors enhance the efficiency of this process by concentrating mRNPs at the nuclear face of the pores. Although this enhancement has been explored mainly with the yeast TREX-2 complex, recent work has indicated that mammalian cells employ GANP (Germinal-centre Associated Nuclear Protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1-containing mRNPs to NPCs. GANP is constructed from several domains that show local homology to FG-nucleoporins, the yeast mRNA export factor Sac3p and the mammalian MCM3 acetyltransferase. Whereas yeast TREX-2 is located primarily at nuclear pores, some GANP is located in the nuclear interior in addition to that found at the pores. GANP depletion inhibits bulk mRNA export, resulting in retention of mRNPs and NXF1 in punctate foci within the nucleoplasm, consistent with GANP's being an integral component of the mammalian mRNA export machinery. Here, we discuss the model for GANP function presented in our recent paper and its implications for the mechanism of mRNA export in mammalian cells.

Cancer cells often contain p53 abnormalities that impair cell-cycle checkpoint progression and cause resistance to various anti-cancer treatments. DNA damage occurs at actively transcribed genes during G1-phase in yeast cells that have a deficient mRNA export capacity. Here, we show that germinal center-associated nuclear protein (GANP), a homologue of yeast Sac3 that is involved in mRNA export, is indispensable for ensuring the stability of human genomic DNA and that GANP knockdown causes apoptosis and necrosis of p53-insufficient cancer cells. Ganp small interfering RNA (siGanp)-induced DNA damage, accompanied by a decrease in the number of cells in S-phase, caused late apoptosis and necrosis in p53-insufficient cancer cells through both caspase-dependent and -independent mechanisms. siGanp effectively induced DNA damage leading to cell death in p53-insufficient cancer cells in vitro and protect the growth of cancer cells transplanted into immunocompromized mice, suggesting that siGanp has potential as a selective treatment for p53-insufficient cancer cells.

The immune system produces specific antibodies (Ab) against any antigens (Ag) of exogenous and endogenous origins with a diverse repertoire of V-region specificities. The primary V-region repertoire is created by the rearrangement of immunoglobulin (Ig) V-region, D- and J-segments with the insertion of N- and P-sequences during early B cell differentiation. Recent studies revealed that secondary diversification of the IgV-region generated in the peripheral lymphoid organs plays a critical role in the generation of effective Ab production for protection from various pathogens. Naïve B cells that react with Ags initiate proliferation and differentiation in the follicular region and create the germinal centres (GCs), where activation-induced cytidine deaminase (AID)-dependent IgV-region somatic hypermutation (SHM) and class-switch recombination generate high-affinity and class-switched mature Ag-specific B cells. Our studies have discovered a 210-kDa nuclear protein, named GC-associated nuclear protein (GANP) that is up-regulated in GC B cells during the T cell-dependent (TD) immune responses. By studying mice with mutant forms of the ganp gene, we demonstrated that GANP is essential for the generation of high-affinity B cells against TD-Ag by affecting SHM at the IgV-regions. GANP is associated with AID in the cytoplasm and the GANP/AID complex is recruited to the nucleus, specifically, the chromatin, and targeted selectively to the IgV-region gene in B cells. GANP augments the access of AID towards IgV-regions in B cells. Here, we review the role of GANP in acquired immunity through the detailed analysis of the molecular mechanism generating SHM specifically at IgV-regions in B cells.

AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C --> U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.

Germinal center-associated nuclear protein (GANP) is a 210-kDa protein that is upregulated in rapidly proliferating B cells. GANP contains regions for RNA-primase and minichromosome maintenance 3 (MCM3)-associated activities, as well as a Sac3-homology region, which is associated with mRNA export in yeast. Here, we examined the role of GANP in mRNA export and cell proliferation in mammalian cells. The ganp small interfering RNA (siRNA) induced cell-cycle arrest at the G2/M-phase, but increased abnormal chromosome alignment of metaphase chromosomes and cell apoptosis in HeLa cells. These changes were not associated with either the abnormality of the spindle assembly checkpoint or the expression level of cohesin. ganp siRNA disrupted the assembly and localization of cohesin at the centromeres in metaphase cells, which is a quite similar phenotype caused by Shugoshin-1 (Sgo1) siRNA-treatment, which was reported previously. ganp siRNA did induce a selective decrease in Sgo1 transcript levels in the cytoplasm, resulting in a lack of cohesin at the centromeres in metaphase and premature separation of the sister chromatids at mitosis. GANP lacking the Sac3-homology region caused the dominant-negative effect with similar abnormalities and impaired mRNA export. Thus, human GANP is critically involved in cell proliferation at the mitotic phase through its selective support of Sgo1 mRNA export.

Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY and SR splicing factors and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)(+)RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.

Malignant glioma (MG) is highly proliferative and invasive, with the malignant characteristics associated with aneuploidy and chromosomal instability (CIN). Here, we found that the level of germinal center-associated nuclear protein (GANP), a mammalian homologue of yeast Sac3, was markedly decreased in MGs with a poor prognosis; and thus we explored the effect of its decrease on cell-cycle progression of MG cell lines. Glioblastomas showed a significantly lower level of ganp mRNA than anaplastic astrocytomas, as measured by real-time reverse transcription-PCR, in 101 cases of adult MG. MGs of ganp(Low) expression displayed more malignant characteristics, with loss of heterozygosity on chromosome 10, epidermal growth factor receptor gene amplification, and significantly poorer prognosis than the ganp(High) group. Human diploid fibroblasts depleted of ganp mRNA by the RNA interference (RNAi) method showed a decreased percentage of S-phase cells and a cellular-senescence phenotype. MG cell lines harboring abnormalities of various cell-cycle checkpoint molecules displayed slippage of mitotic checkpoints and an increased proportion of hyperploid cells after ganp RNAi-treatment. These results suggest that GANP protects cells from cellular senescence caused by DNA damage and that a significant decrease in GANP expression leads to malignancy by generating hyperploidy and CIN.

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.

Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways.

Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae, DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat beta-galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp(+/-) mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sceI restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells.

The senescence-accelerated mouse (SAM) is an animal model for studying senescence and age-associated disorders due to its inherited aging phenotype. The SAM/prone8 (SAMP8) is a useful animal model to investigate the fundamental mechanisms involved in age-related learning and memory deficits that may have relevance to age-associated AD, while SAM/resistant1 (SAMR1) shows normal. To identify genes rendering the cognitive deterioration with aging, the subtractive cDNA libraries containing 1924 clones with the positive ratio of 96.18% were generated and the microarray containing 3136 cDNA was prepared. The results of screening libraries by the microarray showed that of all 91 differentially expressed genes, 50 were over-expressed and 41 were low-expressed in SAMP8. Some of the identified genes were confirmed by the real time quantitative RT-PCR. These results indicated the profiles of gene expression in the hippocampus of SAMP8 and SAMR1 were significantly different, which may play important roles in the age-related cognitive deficit in SAMP8, suggesting those genes related to the cognitive deficient or pathology change in the brain of SAMP8 may be potential gene targets for Alzheimer's disease therapy.

Glucocorticoids are widely used to treat inflammatory diseases but have a number of side effects that partly are connected to inhibition of cell proliferation. Glucocorticoids mediated their action by binding to the glucocorticoid receptor. In the present study, we have identified by two-hybrid screens the germinal center-associated protein (GANP) and MCM3-associated protein (MCM3AP), a splicing variant of GANP, as glucocorticoid receptor interacting proteins. GANP and MCM3AP can bind to the MCM3 protein involved in initiation of DNA replication. Glutathione-S-transferase-pull-down and co-immunoprecipitation assays showed that the C-terminal domain of GANP, encompassing MCM3AP, interacts with the ligand-binding domain of the glucocorticoid receptor. Characterization of the intracellular localization of GANP revealed that GANP is shuttling between the nucleus and the cytoplasm. Furthermore, we show that glucocorticoids are unable to inhibit DNA replication in HeLa cells overexpressing MCM3AP suggesting a role for both glucocorticoid receptor and GANP/MCM3AP in regulating cell proliferation.

Germinal center-associated nuclear protein (GANP) is a newly cloned molecule that is up-regulated in the germinal center B cells. Although GANP functions in the regulation of DNA repair during replication and survival of B cells, little is known about its expression in melanocytic cells.

To investigate whether GANP and phosphorylated-GANP (P-GANP) are expressed in cultured human melanocytes and melanoma cells and in benign and malignant melanocytic lesions. In addition, we aim to determine whether GANP and P-GANP are associated with malignant transformation of melanocytic lineage.

GANP and P-GANP expression in cultured melanocytic cells was analyzed by immunostaining and in vitro kinase assay. GANP and P-GANP expression in melanocytic lesions was analyzed by immunohistochemistry.

GANP and P-GANP were up-regulated in cultured melanoma cells compared to melanocytes. GANP and P-GANP were restricted to nucleus of melanocytes but co-expressed in cytoplasm of melanoma cells. On the other hand, GANP and P-GANP were widely expressed at various levels in melanocytic nevi and melanoma lesions with nuclear and cytoplasmic staining pattern. Melanoma cells showed a stronger intensity of GANP and P-GANP than melanocytic nevus cells, however the staining intensity in primary melanoma lesions was not associated with any clinicopathological variables. Cytoplasmic GANP and P-GANP expression was associated with MCM3 and Ki67 expression.

These data suggest, for the first time, that GANP and P-GANP are up-regulated in cultured melanoma cells compared to melanocytes and also they are widely expressed in benign and malignant melanocytic tumor cells.

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP-/- B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

Lymphomas arise containing abnormalities of various differentiation stage-specific molecules. In the study reported here, we have shown abnormal up-regulation of germinal center B cell-associated GANP in various human lymphomas including mantle cell, diffuse large B cell, and Hodgkin lymphoma, by immunohistochemical analysis. To study the role of GANP in lymphomagenesis, we generated mutant mice (ganp-Tg) that express the transgenic ganp gene under immunoglobulin enhancer and promoter control. Ganp-Tg mice showed a high incidence of lymphomagenesis (29.5%) after aging with a non-B/non-T cell surface phenotype having slight CD45R/B220 expression and Ig transcripts of rearranged VH-DH-JH IgH loci. Lymphomas generated in ganp-Tg mice displayed similar pathologic characteristics to mouse reticulum cell neoplasm or Hodgkin lymphoma-like lesions. The VH sequences of individual mice showed that the tumors proliferated from a single clone or oligoclones, as is found in human diffuse large B-cell lymphomas and Hodgkin lymphoma. These results suggest that GANP overexpression is a causative factor in the generation of B lymphomas.

Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K(D) approximately 2.50 x 10(-7) M) of IgG1/lambda1 and contained the V(H)186.2 region with W33L mutation. Ganp(Tg) generated much higher affinity (K(D) > 1.57 x 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K(D) > 9.90 x 10(-11) M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.

Saccharomyces Sac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to alpha-tubulin, at M phase. RNA interference suppression of endogenous shd1 caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection with shd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated that shd1 is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-kappaB, elongation factor 1alpha, CD79b, octamer binding factor 1, Ig H chain, stathmin, and gamma-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn-->PU.1-->GANP.

The minichromosome maintenance (or MCM) protein family is composed of six related proteins that are conserved in all eukaryotes. They were first identified by genetic screens in yeast and subsequently analyzed in other experimental systems using molecular and biochemical methods. Early data led to the identification of MCMs as central players in the initiation of DNA replication. More recent studies have shown that MCM proteins also function in replication elongation, probably as a DNA helicase. This is consistent with structural analysis showing that the proteins interact together in a heterohexameric ring. However, MCMs are strikingly abundant and far exceed the stoichiometry of replication origins; they are widely distributed on unreplicated chromatin. Analysis of mcm mutant phenotypes and interactions with other factors have now implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure. These experiments indicate that the MCMs are central players in many aspects of genome stability.

Acquired immunity depends on proliferation and differentiation of antigen (Ag)-specific B cells in germinal centers (GCs) of lymphoid follicles in response to T cell-dependent Ags. Here, we studied the function of GC-associated nuclear protein that is selectively up-regulated in GC-B cells. B cell-specific ganp-deficient mice were compromised in affinity maturation of hapten-specific antibodies against T cell-dependent Ags with retarded development of GCs. B cell numbers and development, serum Ig levels, mitogen-induced B cell proliferation in vitro, and responses to T cell-independent Ag were nearly normal; however, the mutant B cells showed a decrease in anti-CD40-induced proliferation and an increased susceptibility to B cell apoptosis in vitro and in vivo. B cell-specific ganp-deficient mice showed a decreased frequency of variable-region somatic mutations, especially of the high-affinity type (W(33) --> L) in the V(H)186.2 region against nitrophenyl-chicken gamma globulin, whereas the class switching was normal. We conclude that GC-associated nuclear protein is necessary for generation or maintenance of B cells with high-affinity B cell Ag receptors during the maturation in GCs.

Autoimmune-prone mice bear a hyper-active B cell population generated spontaneously in peripheral lymphoid organs. Expression of beta RNA-primase GANP was shown to be an activation marker in lymphoid follicle germinal center (GC) B cells after immunization with T cell-dependent antigen (TD-Ag) in normal mice. In this study, we examined the expression of GANP in lymphoid tissues of autoimmune-prone mice. GANP expression was up-regulated in GC-B cells after stimulation with TD-Ags; however, highly GANP-positive (GANP(hi)) cells were also observed in lymph nodes of non-immunized MRL/lpr mice. GANP(hi)cells in lymph nodes as well as in spleens of the different autoimmune-prone strains, MRL/lpr, NZB, (NZBxNZW)F1 and BXSB, gradually increased with age. This population was detected only in small numbers in the red pulp region of the spleen after immunization with TD-Ag in normal C57BL/6 and BALB/c mice. GANP(hi)cells had a B220(-)IgM(+)Syndecan-1(+)phenotype, but were negative for PAS-staining and bromo-deoxyuridine incorporation. These results demonstrate that GANP(hi)plasma-like cells appear in lymph nodes of autoimmune mice during aging, suggesting that the new plasma cell population might be generated after hyper-activation of B cells during the course of autoimmune disease.

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B'/PR61, and B"/PR72). We identified two functional and highly conserved Ca(2+)-binding EF-hand motifs in human B"/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca(2+) to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca(2+) with different affinities. Ca(2+) induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca(2+) on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A(T72)) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca(2+) on PP2A(T72) activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.

Minichromosome maintenance (MCM) proteins are essential components of pre-replication complexes, which limit DNA replication to once per cell cycle. MCM3 acetylating protein, MCM3AP, binds and acetylates MCM3 and inhibits cell cycle progression. In the present study, we examined inhibition of the cell cycle by MCM3AP in a cell-free system. We show here that wild type MCM3AP, but not the acetylase-deficient mutant, inhibits initiation of DNA replication, but not elongation. Both wild type and acetylase-deficient mutant MCM3AP, however, can bind to chromatin through interaction with MCM3. These results indicate that MCM3 acetylase activity of MCM3AP is required to inhibit initiation of DNA replication and that association of MCM3AP to chromatin alone is not sufficient for the inhibition. We also show that interaction between MCM3 and MCM3AP is essential for nuclear localization and chromatin binding of MCM3AP. Furthermore, the chromatin binding of MCM3AP is temporally correlated with that of endogenous MCM3 when cells were released from mitosis. Hence, MCM3AP is a potent natural inhibitor of the initiation of DNA replication whose action is mediated by interaction with MCM3.

Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs. Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p. In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation. In addition, Sac3p binds to the mRNA exporter Mex67p-Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC). Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects. Taken together, our data suggest that the novel Sac3p-Thp1p complex functions by docking the mRNP to specific nucleoporins at the nuclear entrance of the NPC.

GANP, carrying DNA-primase and MCM3-binding domains, is up-regulated in germinal centre B cells. To understand the regulatory function of GANP upon MCM complex, we searched for GANP-associated molecules by yeast two-hybrid screening.

Using the 1 kb fragment (G5) of the ganp cDNA, we identified a clone named G5PR that is structurally homologous to known regulatory subunits of protein phosphatases (PPases) and determined the association of G5PR with GANP in vivo in the DNA transfectant. G5PR is associated with protein phosphatase 5 (PP5) through its tetratricopeptide-repeat (TPR) domain. Pull-down assays demonstrated that G5PR is also associated with protein phosphatase 2A (PP2A), the complex of A subunit (PR65) and the catalytic (C) subunit (PP2Ac), similar to the B" subunit. The G5PR-associated complex had phosphatase activity on casein, histone H1 and MCM3 in vitro, but the addition of G5PR did not stimulate or inhibit the phosphatase activities of PP5 and PP2A. The cellular localization of G5PR in transfected cells varies during cell cycling, appearing in the nucleus during prophase, in the peri-chromatin during mitotic phase, and in the cytoplasm after cell division.

G5PR is capable of recruiting two kinds of PPases, PP5 and PP2A, into the GANP/MCM3 complex, which might regulate its phosphorylation state during cell cycle progression.

Germinal center-associated DNA primase (GANP) associated with MCM3 of the DNA replication complex is up-regulated selectively in germinal center B cells. We studied promoter activity of the 5' region involved in the developmental stage-dependent expression in B lineage cells by luciferase reporter assay. Selective regulation of ganp expression was observed in the -737-bp promoter region in B and plasma cell lines but was significantly low in pre-B and T cell lines. The deletion constructs displayed a gap decrease after shortening the region from -134 to -108 bp. Further narrowing suggested the involvement of the PU.1 consensus sequence at -126 bp by electrophoretic mobility shift assay. The protein component PU.1 complex is not inhibited with mutated probes at the consensus site but is inhibited with the known PU.1 probe of CD72 and with anti-PU.1 antibody. Moreover, introduction of PU.1 cDNA enhanced the reporter gene activity in a dose-dependent manner in B cells, whereas the reporter construct with the mutated PU.1 site did not respond. Anti-CD40 stimulation induced the reporter activity with a 100% increase, which is not observed with the PU.1-mutated reporter construct. These results demonstrate that the germinal center-associated DNA primase expression is partly regulated by the transcription factor PU.1 expressed in B lineage cells.