Atsuko Masumi

An IFN-γ-Inducible Transcription Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates IL-12 p40 Expression in Macrophages

IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-γ/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-γ/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP−/− mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-γ/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP−/− cells, even in the absence of IFN-γ/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-γ/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-γ and LPS. Taken together, our results show that ICSBP induced by IFN-γ/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.

The Histone Acetylase PCAF Is a Phorbol-Ester-Inducible Coactivator of the IRF Family That Confers Enhanced Interferon Responsiveness

ABSTRACT Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-α-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-α-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness.

IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages

IRF‐8/ICSBP and IRF‐1 are IRF family members whose expression is induced in response to IFN‐γ in macrophages. IL‐12 is a cytokine produced in macrophages that plays a critical role in host defense. IFN‐γ and bacterial lipopolysaccharide (LPS) induce IL‐12p40 transcription, which is necessary for the production of IL‐12. We have previously shown that IL‐12p40 expression is impaired in ICSBP‐deficient mice and that transfection of ICSBP together with IRF‐1 can activate IL‐12p40 expression in mouse macrophage cells. To further study the role of ICSBP and IRF‐1, we investigated murine IL‐12p40 promoter activity in the macrophage cell line RAW 264.7. We show here that co‐transfection of ICSBP and IRF‐1 synergistically stimulates IL‐12 promoter activity to a level comparable to that induced by IFN‐γ/LPS. Mutation of the Ets or NFκB site previously shown to be important for IL‐12p40 transcription did not abolish the activation by ICSBP and IRF‐1. However, mutation of the ISRE‐like site found downstream from the NFκB and C/EBP sites abrogated the activation by ICSBP and IRF‐1. Together, these results indicate that ICSBP and IRF‐1 cooperatively stimulate murine IL‐12 transcription through a novel regulatory element in the murine promoter.

Identification of cancer stem cells in a Tax-transgenic (Tax-Tg) mouse model of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 10(4) SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38(-)/CD71(-)/CD117(+) cells (0.03%). Whereas lymphoma did not develop after transplantation of 10(2) SLCs, 10(2) CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117(+) CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model.

Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

ABSTRACT Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation

We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated in a cell growth-dependent manner, which enables it to contribute to the transcription of cell growth-regulated promoters. To clarify the function of acetylation of IRF-2, we investigated the proteins that associate with acetylated IRF-2. In 293T cells, the transfection of p300/CBP-associated factor (PCAF) enhanced the acetylation of IRF-2. In cells transfected with both IRF-2 and PCAF, IRF-2 associated with endogenous nucleolin, while in contrast, minimal association was observed when IRF-2 was transfected with a PCAF histone acetyl transferase (HAT) deletion mutant. In a pull-down experiment using stable transfectants, acetylation-defective mutant IRF-2 (IRF-2K75R) recruited nucleolin to a much lesser extent than wild-type IRF-2, suggesting that nucleolin preferentially associates with acetylated IRF-2. Nucleolin in the presence of PCAF enhanced IRF-2-dependent H4 promoter activity in NIH3T3 cells. Nucleolin knock-down using siRNA reduced the IRF-2/PCAF-mediated promoter activity. Chromatin immunoprecipitation analysis indicated that PCAF transfection increased nucleolin binding to IRF-2 bound to the H4 promoter. We conclude that nucleolin is recruited to acetylated IRF-2, thereby contributing to gene regulation crucial for the control of cell growth.

Application of DNA microarray technology to influenza A/Vietnam/1194/2004 (H5N1) vaccine safety evaluation

We propose that DNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that DNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv.

Peripheral B Cells May Serve as a Reservoir for Persistent Hepatitis C Virus Infection

A recent study by our group indicated that peripheral B cells in chronic hepatitis C (CHC) patients are infected with hepatitis C virus (HCV). This raised the logical question of how HCV circumvents the antiviral immune responses of B cells. Because type I interferon (IFN) plays a critical role in the innate antiviral immune response, IFNβ expression levels in peripheral B cells from CHC patients were analyzed, and these levels were found to be comparable to those in normal B cells, which suggested that HCV infection failed to trigger antiviral immune responses in B cells. Sensing mechanisms for invading viruses in host immune cells involve Toll-like receptor-mediated and retinoic acid-inducible gene-I (RIG-I)-mediated pathways. Both pathways culminate in IFN regulatory factor-3 (IRF-3) translocation into the nucleus for IFNβ gene transcription. Although the expression levels of RIG-I and its adaptor molecule, IFN promoter-stimulator-1, were substantially enhanced in CHC B cells, dimerization and subsequent nuclear translocation of IRF-3 were not detectable. TANK-binding kinase-1 (TBK1) and IĸB kinase Ε (IKKΕ) are essential for IRF-3 phosphorylation. Constitutive expression of both kinases was markedly enhanced in CHC B cells. However, reduced expression of heat shock protein of 90 kDa, a TBK1 stabilizer, and enhanced expression of SIKE, an IKKΕ suppressor, were observed in CHC B cells, which might suppress the kinase activity of TBK1/IKKΕ for IRF-3 phosphorylation. In addition, the expression of vesicle-associated membrane protein-associated protein-C, a putative inhibitor of HCV replication, was negligible in B cells. These results strongly suggest that HCV utilizes B cells as a reservoir for persistent infection.

Deficient RPS19 protein production induces cell cycle arrest in erythroid progenitor cells.

The gene encoding ribosomal protein S19 (RPS19) is one of the responsible genes for Diamond‐Blackfan anaemia (DBA), a congenital erythroblastopenia. Although haplo‐insufficiency of RPS19 has been suggested to be the onset mechanism underlying the pathogenesis of DBA, the sequential mechanism has not been elucidated. In order to analyse the consequences of the missense mutation of RPS19 specific for DBA patients, we made mutated RPS19 expression vectors. Twelve C‐terminally Flag‐tagged missense mutants were exogenously expressed from retroviral vectors and analysed by Western blot analysis and flow cytometry. When these 12 mutants were expressed in the erythro‐leukaemic cell lines K562 and human bone marrow CD34+ cells, almost all of the mutant proteins (except for G120R) were unstable, and the levels of mutated RPS19 protein were significantly low. To address the effect of deficient RPS19 expression on cell proliferation, RPS19 was downregulated by siRNA. Repressive expression of RPS19 in human CD34+ cells produced an elevated number of cells at G0 and induced erythroid progenitor‐specific defects in BM cells. These results suggest that abnormal ribosomal biogenesis causes inadequate cell cycle arrest in haematopoietic progenitors, and that, subsequently, erythroid progenitors are specifically hampered. These in vitro phenotypes of genetically manipulated CD34+ cells mimic DBA pathogenesis.