Biomedical subjects

Fumiko Hirose

Publications and source records attributed to Fumiko Hirose.

At least 19 recordsLinked to original sources

PubMed2007

hDREF regulates cell proliferation and expression of ribosomal protein genes.

Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G(1) to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

Base Sequence↗

PubMed2007

Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding.

We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.

Active Transport, Cell Nucleus↗

PubMed2006

Orphan nuclear receptor Nur77 accelerates the initial phase of adipocyte differentiation in 3T3-L1 cells by promoting mitotic clonal expansion.

Nur77 is an orphan member of the nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. Although Nur77 mRNA is induced in the early stage of adipogenesis of 3T3-L1 cells, its roles are not known. To address this issue, we closely inspected the expression of Nur77 mRNA and protein during differentiation of 3T3-L1 cells. Nur77 was induced rapidly and transiently at both mRNA and protein levels only in the initial phase of differentiation induction, and localized almost exclusively in the nuclei. Isobutylmethylxanthine was essential for the induction of Nur77 protein, acting by at least in part protecting the protein from rapid degradation by proteasome. Nur77 siRNA resulted in delayed adipogenesis in 3T3-L1, accompanied by retarded mitotic clonal expansion. These effects were mediated at least partly by decreased expression of cyclins D and E. Constitutive expression of Nur77 inhibited adipogenesis of 3T3-L1, accompanied by enhanced expression of cyclin D1 and prolonged mitotic clonal expansion. Moreover, constitutive expression of Nur77 inhibited, but transient induction of Nur77 promoted, adipogenesis in NIH-3T3 cells. These results suggest that Nur77 accelerates adipocyte differentiation by regulating cell cycle progression and the rapid and transient induction is crucial for its action.

3T3-L1 Cells↗

PubMed2006

MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor alpha.

Cytosolic lipid droplets (LDs) are multifunctional organelles that exist in all types of eukaryotic cells and control lipid homeostasis. In mammalian cells LDs contain a class of proteins in their surface layers that share a homologous sequence called the PAT domain, including perilipin, adipose differentiation-related protein (ADRP), a tail-interacting protein of 47 kDa (TIP47), and S3-12, which are distributed tissue- or cell type-selectively. Expression in some cases is regulated by peroxisome proliferator-activated receptors (PPARs). In this study we identified a new PAT family member named MLDP (myocardial LD protein) in a murine cDNA data base and showed the mRNA and protein to be highly enriched in the heart and also expressed at lower levels in the liver and adrenals. Upon subcellular fractionation, a substantial amount of MLDP was detected in the top fraction enriched with LDs. Furthermore, overexpressed MLDP tagged with green fluorescent protein accumulated at the surfaces of LDs and co-localized with perilipin and ADRP. Deletion analysis demonstrated the N-terminal region containing a PAT-1 domain and the following 33-mer domain to be required for targeting of MLDP to LDs. MLDP was found to be up-regulated at both mRNA and protein levels in the heart and liver by a selective ligand for PPARalpha, Wy14,643, but not in PPARalpha knock-out mice. MLDP expression was also increased upon fasting in parallel with ADRP. These results indicate that MLDP is a bona fide new PAT family member localized in LDs. Its expression depends on the physiological conditions and the action of PPARalpha.

3T3-L1 Cells↗

PubMed2006

Aspects of the regulatory mechanisms of PPAR functions: analysis of a bidirectional response element and regulation by sumoylation.

Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear receptor superfamily. A wide variety of compounds including hypolipidemic agents, antidiabetic drugs, and long-chain fatty acids are the potential ligands of PPARs. To approach the regulatory mechanisms of PPARs, we studied on two subjects in this work. First, we identified a functional PPAR-binding site in the spacer region between the PEX11alpha and perilipin genes, which are arranged in tandem on the mouse genome. By gene reporter assays and in vivo as well as in vitro binding assays, we show that these genes are regulated tissue-selectively through this common binding site: The PEX11alpha gene is activated by PPARalpha in the liver, whereas the perilipin gene by PPARgamma in the adipose tissue. As the second subject, we found that PPARgamma2 is conjugated with small ubiquitin-related modifier (SUMO) at a specific lysine residue in the amino-terminal region. By site-directed mutagenesis combined with gene reporter assays and sumoylation analyses, we show that sumoylation represses the ligand-independent transactivating function carried by this region, and hence negatively regulates the whole transactivating competence of PPARgamma2. In addition, phosphorylation at a specific site in the amino-terminal region represses the transactivation by PPARgamma2 possibly through enhancing sumoylation.

Adipose Tissue↗

PubMed2006

Peroxisome proliferator-activated receptor subtypes differentially cooperate with other transcription factors in selective transactivation of the perilipin/PEX11 alpha gene pair.

Perilipin is an adipocyte-specific protein associated with lipid droplets that is crucial for the regulation of storage and mobilization of lipids. We earlier reported that the mouse perilipin gene is regulated by peroxisome proliferator-activated receptor (PPAR) gamma through a peroxisome proliferator-response element (PPRE) positioned upstream of the perilipin promoter. Moreover, we showed that this PPRE also controls expression of the PEX11alpha gene, which is located further upstream. We show here that three elements, A, B, and C, in close proximity downstream of the PPRE, are essential for transactivation of the perilipin gene by PPARgamma. Electrophoretic gel-mobility shift assays demonstrated that nuclear factor (NF)-1 subtypes bind specifically to element B. Furthermore, chromatin immunoprecipitation using 3T3-L1 cells revealed that NF-1A and NF-1B bind to element B in a differentiation-dependent fashion, whereas binding is constitutive with NF-1C and NF-1X. Element C is likely to be a binding motif for nuclear receptors. With PPARalpha, elements A-C do not appear to be required for transactivation of the PEX11alpha gene, so that cooperation with other transcription factors may be differentially involved in selective transactivation of the PEX11alpha and perilipin genes by different PPAR subtypes.

Animals↗

PubMed2005

Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway.

DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase alpha in Drosophila. We have previously reported that overexpression of DREF in developing eye imaginal discs induced ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, we have carried out a screen for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (-205 to -198), and DRE3 (-709 to -702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway.

5' Flanking Region↗

PubMed2005

GDNF-inducible zinc finger protein 1 is a sequence-specific transcriptional repressor that binds to the HOXA10 gene regulatory region.

The RET tyrosine kinase receptor and its ligand, glial cell line-derived neurotrophic factor (GDNF) are critical regulators of renal and neural development. It has been demonstrated that RET activates a variety of downstream signaling cascades, including the RAS/mitogen-activated protein kinase and phosphatidylinositol-3-kinase(PI3-K)/AKT pathways. However, nuclear targets specific to RET-triggered signaling still remain elusive. We have previously identified a novel zinc finger protein, GZF1, whose expression is induced during GDNF/RET signaling and may play a role in renal branching morphogenesis. Here, we report the DNA binding property of GZF1 and its potential target gene. Using the cyclic amplification and selection of targets technique, the consensus DNA sequence to which GZF1 binds was determined. This sequence was found in the 5' regulatory region of the HOXA10 gene. Electrophoretic mobility shift assay revealed that GZF1 specifically binds to the determined consensus sequence and suppresses transcription of the luciferase gene from the HOXA10 gene regulatory element. These findings thus suggest that GZF1 may regulate the spatial and temporal expression of the HOXA10 gene which plays a role in morphogenesis.

Animals↗

PubMed2005

Negative regulation of adipogenesis from human mesenchymal stem cells by Jun N-terminal kinase.

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes, osteoblasts, and chondrocytes, under appropriate culture conditions. We found that SP600125, an inhibitor of Jun N-terminal kinase (JNK), promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors, CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2, which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus, JNK represses adipogenesis from hMSCs probably by, at least in part, inhibiting the transactivating function of CRE-binding protein. Another action of JNK, phosphorylation at Ser(307) of insulin receptor substrate-1, was also predicted to contribute to the repression of adipogenesis.

Adipocytes↗

PubMed2004

Armadillo/Pangolin regulates PCNA and DREF promoter activities.

Here we show that Armadillo and Pangolin (dTCF), downstream effectors of the Wingless (Wg) signal transduction pathway, activate transcription of the important DNA replication-related genes encoding Drosophila proliferating cell nuclear antigen (PCNA) and DNA replication-related element-binding factor (DREF). By transient luciferase expression assays and band mobility shift assays, we demonstrated the PCNA gene to be a direct target gene for the Armadillo/Pangolin complex. Using a GAL4-UAS system, stimulation of the PCNA gene by Armadillo/Pangolin was confirmed in adult females. From the published reports of an inhibitory role, we expected that Drosophila CREB-binding protein (dCBP) would interfere with activation. However, effects were only observed with the DREF but not the PCNA gene. In the latter case, as in mammals, dCBP could potentiate Armadillo-mediated activation. These results suggest that first, PCNA and DREF genes are targets of the Armadillo/Pangolin complex and second, dCBP modulates Wg signaling in a gene-specific manner.

Animals↗

PubMed2004

Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila.

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

Alkylating Agents↗

PubMed2004

Genetic link between p53 and genes required for formation of the zonula adherens junction.

Ectopic expression of human p53 in Drosophila eye imaginal disc cells induces apoptosis and results in a rough eye phenotype in the adult flies. We have screened Drosophila stocks to identify mutations that enhance or suppress the p53-induced rough eye phenotype. One of the dominant enhancers of the p53-induced rough eye phenotype corresponds to a loss-of-function mutation of the crumbs gene, which is essential for the biogenesis of the zonula adherens junction and the establishment of apical polarity in epithelial cells. Enhancement of p53-induced apoptosis in the eye imaginal discs by a half-reduction of the crumbs gene dose was confirmed by a TUNEL method. Furthermore, mutations of genes for Shotgun (Drosophila E-cadherin) and Armadillo (Drosophila beta-catenin), the two main components of the adherens junction, also strongly enhanced the p53-induced rough eye phenotype. These results suggest that human p53 senses subtle abnormality at the adherens junction or in signals derived from the junction, and consequently induces apoptosis to remove abnormal cells from tissue. Thus p53 likely plays a role as a guardian of the tissue not only by sensing the damaged DNA, but also by sensing signals from the adherens junction.

Adherens Junctions↗

PubMed2004

DREF is required for EGFR signalling during Drosophila wing vein development.

The DNA replication-related element binding factor (DREF) has been suggested as being involved in regulation of DNA replication- and proliferation-related genes in Drosophila. Recently, by searching the Drosophila genome database, we also found DRE-like sequences in the 5'-flanking regions of many genes with other functions. In addition, immunostaining of polytene chromosomes with an anti-DREF monoclonal antibody revealed that DREF can bind to a hundred regions of polytene chromosomes, suggesting regulation of multiple genes and multiple roles in vivo. When we over-expressed DREF protein or inverted repeat RNA of the DREF gene in wing imaginal discs using the GAL4-UAS targeted expression system in Drosophila, the results were veins of increased width and a loss of veins, respectively. With DREF over-expression, Rolled, a Drosophila MAPK homologue, was ectopically activated. Furthermore, half reduction of the D-raf gene dose suppressed this DREF-induced vein of increased width phenotype. In addition, when DREF transcripts were reduced by introducing double-stranded RNA of the DREF gene into S2 cells, the D-raf gene promoter activity was diminished to 4%. These data indicate that DREF is involved in regulation of vein formation through the activation of EGFR signalling in the Drosophila wing imaginal discs.

Animals↗

PubMed2004

The transactivating function of peroxisome proliferator-activated receptor gamma is negatively regulated by SUMO conjugation in the amino-terminal domain.

Peroxisome proliferator-activated receptor (PPAR)-gamma2, a member of the nuclear hormone receptor superfamily, plays a key role in adipocyte differentiation. Its amino-terminal region carries a ligand-independent gene-activating function, AF-1, and is composed of activation as well as repression domains. We have found PPARgamma2 and its isoform, PPARgamma1, to be modified by small ubiquitin-related modifier (SUMO)-1 in vivo, at a lysine residue in the repression domain. In reporter assays, a sumoylation-defective K107R mutant of PPARgamma2 exhibited much stronger transactivation than the wild-type, comparable with that of a mutant deleted for the repression domain. A close inverse correlation was observed between the levels of sumoylation and transactivation by PPARgamma2, in analyses employing PPARgamma2 forms with mutations in the sumoylation motif and a dominant-negative mutant of the SUMO conjugating enzyme, Ubc9. Studies with phosphorylation-defective mutants suggested that phosphorylation at S112 of PPARgamma2 promotes K107 sumoylation, and this latter exerts the more potent repressive effects. The K107R mutant PPARgamma2, when infected into NIH3T3 cells with a viral vector, promoted differentiation into adipocytes more efficiently than the wild-type. These observations provide evidence that sumoylation is involved in negative regulation of the transactivating function of PPARgamma2.

HeLa Cells↗

PubMed2003

Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila melanogaster and virilis.

A DNA replication-related element (DRE)-binding factor (DREF) has been revealed to be an important transcription factor for activating promoters of cell proliferation and differentiation related genes. The amino acid sequences of DREF are conserved in evolutionary separate Drosophila species, Drosophila melanogaster (Dm) and Drosophila virilis (Dv) in three regions. In the present study, evidence was obtained that there are several highly conserved regions in the 5' flanking region between the DmDREF and DvDREF genes. Band mobility shift assays using oligonucleotides corresponding to these conserved regions revealed that specific trans-acting factors can bind to at least three regions -554 to -543 (5'-TTTGTTCTTGCG), -81 to -70 (5'-GCCCACGTGGCT) and +225 to +234 (5'-GCAATCAGTG). Using a transient luciferase expression assay, we demonstrated that the region -554 to -543 functions as a negative regulatory element for DmDREF promoter activity, while the regions -77 to -70 (5'-ACGTGGCT) and +225 to +236 (5'-GCAATCAGTGTT) function as positive regulatory elements. In previous studies, we observed that expression of the homeodomain protein Zerknüllt (Zen) represses PCNA gene transcription, by reducing the DNA binding activity of DREF. Here we show Zen downregulates DREF gene promoter activity through action on the region between +241 and +254 (5'-AGAATACTCAACA). In addition, the DmDREF promoter contains five DREs. Using a double stranded RNA-mediated interference method, we generated evidence that expression of DmDREF could be auto-regulated by DREF through the third DRE located at +211 to +218. In living flies we obtained results consistent with those obtained in vitro and in cultured cells. The study thus indicates that DmDREF is effectively regulated via highly conserved regions between the DmDREF and DvDREF promoters, suggesting the existence of common regulatory factors, and that DmDREF can be positively regulated by itself via the third DRE located in its most highly conserved region.

5' Flanking Region↗

PubMed2003

Identification of a human homologue of the DREF transcription factor with a potential role in regulation of the histone H1 gene.

A human homologue (hDREF/KIAA0785) of Drosophila DREF, a transcriptional regulatory factor required for expression of genes involved in DNA replication and cell proliferation, was identified by BLAST search. Amino acid sequences corresponding to three regions highly conserved between two Drosophila species also proved to be very similar in the hDREF/KIAA0785 polypeptide. A consensus binding sequence (5'-TGTCG(C/T)GA(C/T)A) for hDREF/KIAA0785, determined by the CASTing method, overlapped with that for the Drosophila DREF (5'-TGTCGATA). We found hDREF/KIAA0785 binding sequences in the promoter regions of human genes related to cell proliferation. Analyses using a specific antibody revealed that an hDREF/KIAA0785 binds to the promoter region of the histone H1 gene. Co-transfection experiments with an hDREF/KIAA0785-expressing plasmid and a histone H1 promoter-directed luciferase reporter plasmid in HeLa cells revealed possible activation of the histone H1 promoter. Immunohistochemical analysis demonstrated that hDREF/KIAA0785 is localized in the nuclei. Although the expression level of the factor was found to be low in serum-deprived human normal fibroblasts, the amount was increased by adding serum to cultures and reached a maximum during S phase. RNA interference experiments targeting hDREF/KIAA0785 resulted in inhibition of S phase entry and reduction of histone H1 mRNA in HeLa cells. These results suggest that expression of hDREF/KIAA0785 may have a role in regulation of human genes related to cell proliferation.

Amino Acid Sequence↗

PubMed2003

Spatio-temporal expression of Drosophila mitochondrial transcription factor A during development.

We previously reported cloning of the Drosophila mitochondrial transcription factor A (D-mtTFA) gene, and characterization of a recombinant protein. In this report, the expression and distribution patterns of D-mtTFA during development are described. D-mtTFA mRNA and its protein product were found to be abundant in cells of tissues undergoing both proliferation and polytenization. Furthermore, a DRE-like sequence present in the D-mtTFA gene showed promotor activity in Drosophila Kc cells. In addition, D-mtTFA was detected in brain throughout the developmental process, as well as in non-proliferating tissues, such as flight muscle and cardia, and was also found in spermatids of imagos. D-mtTFA possesses a nuclear-targeting sequence, and is present in the nucleus at the syncytial stage, where bundles of 64 spermatids are present during spermatogenesis. The results suggest that D-mtTFA not only contributes to the mitochondrial DNA transcription and replication system, but also has a role in cell proliferation and development, including spermatogenesis.

Animals↗

PubMed2002

Drosophila damage-specific DNA-binding protein 1 (D-DDB1) is controlled by the DRE/DREF system.

We succeeded in cloning the gene, termed d-ddb1, for a Drosophila homolog of the p127 subunit of the human damage-specific DNA-binding protein, thought to recognize (6-4) photoproducts and related structures. In Drosophila, the gene product (D-DDB1) also appeared to play a role as a repair factor, d-ddb1 knockout Kc cells generated with a RNAi method being sensitive to UV. In addition, UV or methyl methanesulfonate treatment increased d-ddb1 transcripts. However, we found that the gene is controlled by the DRE/DREF system, which is generally responsible for activating the promoters of proliferation-related genes. Moreover, during Drosophila development, the transcription of d-ddb1 changed greatly, with the highest levels in unfertilized eggs, indicating that external injury to DNA is not essential to D-DDB1 function. Interestingly, as with UV irradiation-induced transfer of D-DDB1 to the nucleus from the cytoplasm, during spermatogenesis the protein transiently shifted from one cell compartment to the other. The results indicate that D-DDB1 not only contributes to the DNA repair system, but also has a role in cell proliferation and development.

5' Flanking Region↗