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Biomedical subjects

Fuyuki Ishikawa

Publications and source records attributed to Fuyuki Ishikawa.

At least 19 recordsLinked to original sources
PubMed2006

Tel2 is required for activation of the Mrc1-mediated replication checkpoint.

Proteins belonging to the Tel2/Rad-5/Clk-2 family are conserved among eukaryotes and are involved in various cellular processes, such as cell proliferation, telomere maintenance, the biological clock, and the DNA damage checkpoint. However, the molecular mechanisms underlying the functions of these molecules remain largely unclear. Here we report that in the fission yeast, Schizosaccharomyces pombe, Tel2 is required for efficient phosphorylation of Mrc1, a mediator of DNA replication checkpoint signaling, and for activation of Cds1, a replication checkpoint kinase, when DNA replication is blocked by hydroxyurea. In fact, Tel2 is required for survival of replication fork arrest and for the replication checkpoint in cells lacking Chk1, another checkpoint kinase the role of which overlaps that of Cds1 in cell cycle arrest by replication block. In addition, Tel2 plays important roles in entry into S phase and in genome stability. Tel2 is essential for vegetative cell growth, and the tel2Delta strain accumulated cells with 1C DNA content after germination. In the absence of hydroxyurea, Tel2 is vital in the mutant lacking Swi1, a component of the replication fork protection complex, and multiple Rad22 DNA repair foci were frequently observed in Tel2-repressed swi1Delta cells especially at S phase. In contrast, the cds1Deltaswi1Delta mutant did not show such lethality. These results indicate that S. pombe Tel2 plays important roles in the Mrc1-mediated replication checkpoint as well as in the Cds1-independent regulation of genome integrity.

Cell Cycle Proteins↗
PubMed2006

Loss of linker histone H1 in cellular senescence.

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)-tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA-binding proteins.

Bromodeoxyuridine↗
PubMed2006

Cell-cycle-dependent regulation of telomere binding proteins: roles of Polo-like kinase.

The telomere is a functional complex at chromosomal termini consisting of repetitive DNA and associated proteins, and protects the ends against degradation and fusion. Telomeric repeat binding factors TRF1 and TRF2 bind directly to double-stranded telomeric DNA. Although structurally related, TRF1 and TRF2 contribute to telomere maintenance in distinct ways: TRF1 negatively regulates telomerase-dependent telomere lengthening, whereas TRF2 plays an important role in protecting chromosomal ends. It is not known how the proteinaceous complex manages DNA metabolism such as DNA replication, which requires the recruitment of numerous trans-acting factors. We have found that Xenopus TRF1 (xTRF1) specifically associates with mitotic chromatin and dissociates from interphase replicating chromatin. In contrast, Xenopus TRF2 (xTRF2) binds to telomeric DNA throughout the cell cycle. Interestingly, telomerase activity is associated with the interphase chromatin, but not with the mitotic chromatin. These results support a model in which telomeres form a semi-open configuration that allows access of telomerase and replication machineries, yet protects the chromosomal ends in S phase. Interestingly, M phase specific telomere binding of xTRF1 requires Polo-like kinase, a key regulator of mitosis. We discuss the relevance of our studies and recent findings of other groups to indicate the possible role of Polo-like kinase in telomere regulation.

Animals↗
PubMed2006

Cell-cycle-dependent Xenopus TRF1 recruitment to telomere chromatin regulated by Polo-like kinase.

Telomeres are regulated by a homeostatic mechanism that includes telomerase and telomeric repeat binding proteins, TRF1 and TRF2. Recently, it has been hypothesized that telomeres assume distinct configurations in a cell-cycle-dependent manner, although direct biochemical evidence is lacking. Here we demonstrated that Xenopus TRF1 (xTRF1) associates with telomere chromatin specifically in mitotic Xenopus egg extracts, and dissociates from it upon mitotic exit. Both the N-terminal TRF-homology (TRFH) domain and the linker region connecting the TRFH domain and the C-terminal Myb domain are required for this cell-cycle-dependent association of xTRF1 with chromatin. In contrast, Xenopus TRF2 (xTRF2) associates with chromatin throughout the cell cycle. We showed that Polo-like kinase (Plx1) phosphorylates xTRF1 in vitro. Moreover, the mitotic xTRF1-chromatin association was significantly impaired when Plx1 was immunodepleted from the extracts. Finally, high telomerase activities were detected in association with replicating interphase chromatin compared with mitotic chromatin. These results indicate that telomere chromatin is actively regulated by cell-cycle-dependent processes, and provide an insight for understanding how telomeres undergo DNA metabolisms during the cell cycle.

Animals↗
PubMed2006

Cellular senescence as a stress response.

PURPOSE: This review discusses cellular senescence, the state in which normal cells do not respond to growth stimuli, and shows characteristic alterations in their cytologic and biochemical properties and their gene expression profiles. METHODS AND RESULTS: Cellular senescence is elicited by various stresses. It was recently shown that the stress-induced mitogen-activated protein kinase p38 has a pivotal role in inducing cellular senescence. This finding provided biochemical evidence to support the notion that cellular senescence is a kind of stress response. CONCLUSION: Stress responses are typically found in cells and organisms surviving unfavorable environmental conditions. It can be argued that cellular senescence is an adaptive process that maintains the cell's viability by reducing the energy consumed for reproduction (ie, cell division) and differentiation-related activities.

Cellular Senescence↗
PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗
PubMed2005

Telomere binding protein Taz1 establishes Swi6 heterochromatin independently of RNAi at telomeres.

BACKGROUND: The telomere is a specialized heterochromatin conserved among eukaryotes. However, it remains unknown how heterochromatin protein 1 (HP1) is recruited to telomeres and how telomere heterochromatin is formed. In fission yeast, the RNAi (RNA interference)-RITS (RNA-induced initiation of transcriptional silencing) pathway initiates heterochromatin formation at the centromeres and the silent mat locus by using common DNA sequences, the dg and dh repeats, as the templates for small interfering RNA (siRNA). RESULTS: We found that telomeric repeats are sufficient for the establishment of Swi6 (a fission-yeast HP1 homolog) heterochromatin, and the establishment requires Taz1, a telomere binding protein of the TRF family. Additionally, Swi6 heterochromatin is established by a part of the subtelomere that contains sequences highly homologous to that of the dh repeat, and it is strikingly destabilized by the deletion of both Taz1 and RNAi-RITS. Transcripts from the telomeric dh-homologous region were specifically associated with RITS, and deletion of the telomeric dh-homologous region showed the phenotype similar to that of the rnai mutant in terms of the telomeric silencing, indicating that the RNAi-RITS pathway acts at the telomeric dh-homologous region to establish Swi6 heterochromatin. Furthermore, we found that Taz1 establishes Swi6 heterochromatin independently of the telomeric repeats and the RNAi-RITS pathway at the subtelomeres. CONCLUSION: The telomere heterochromatin is regulated by at least two factors: One is Taz1, which is telomere specific, and the other is RNAi-RITS, which is commonly used at the constitutive heterochromatin regions.

Chromatin Immunoprecipitation↗
PubMed2005

Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D.

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG)n, the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3'-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase.

Circular Dichroism↗
PubMed2005

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha.

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.

Animals↗
PubMed2004

Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells.

Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.

Animals↗
PubMed2004

Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body.

Telomere maintenance is essential for continued cell proliferation. Although most cells accomplish this by activating telomerase, a subset of immortalized tumors and cell lines do so in a telomerase-independent manner, a process called alternative lengthening of telomeres (ALT). DNA recombination has been shown to be involved in ALT, but the precise mechanisms remain unknown. A fraction of cells in a given ALT population contain a unique nuclear structure called APB (ALT-associated promyelocytic leukemia (PML) body), which is characterized by the presence of telomeric DNA in the PML body. Here we describe that hRad9, hHus1, and hRad1, which form a DNA clamp complex that is associated with DNA damage, as well as its clamp loader, hRad17, are constitutive components of APB. Phosphorylated histone H2AX (gamma-H2AX), a molecular marker of double-strand breaks (DSBs), also colocalizes with some APBs. The results suggest that telomeric DNAs at APBs are recognized as DSBs. PML staining and fluorescence in situ hybridization analyses of mitotic ALT cells revealed that telomeric DNAs present at APBs are of both extrachromosomal and native telomere origins. Furthermore, we demonstrated that DNA synthesis occurs at APBs and is significantly inhibited by caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases. Taken together, we suggest that telomeric DNAs at APBs are recognized and processed as DSBs, leading to telomeric DNA synthesis and thereby contributing to telomere maintenance in ALT cells.

Bromodeoxyuridine↗
PubMed2004

Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats.

Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.

Cell Cycle↗
PubMed2004

Ninth Japanese-German Workshop on Molecular and Cellular Aspects of Carcinogenesis, Essen, Germany, 18-20 September, 2003.

The Japanese-German Workshops "Molecular and Cellular Aspects of Carcinogenesis," held biennially since 1987, have been organized traditionally at the University of Essen Medical School and West German Cancer Center Essen in Germany. This 9th Workshop was held on September 18-20, 2003. It was generously supported by the Cancer Research Program of the Ministry of Education, Culture, Sports, Science and Technology and the International Cooperation Program for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control of the Ministry of Health, Labour and Welfare on the Japanese side, and by the Deutsche Forschungsgemeinschaft (DFG) on the German side. Additional support from many other sponsors in Germany and Japan is gratefully acknowledged. The Workshop participants are listed at the end of this Report.

Animals↗
PubMed2004

Molecular mechanisms of DNA end-loop formation by TRF2.

In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher-order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, approximately 1.8 (kb) with different end-structures and observed their higher-order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple-binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA-end structure (blunt, 3'-overhanging, telomeric, non-telomeric). However, when the DNA end carried the telomeric-3'-overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2-mediated DNA loop formation is independent of the end-structure and the 3'-overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end-loop formation by TRFs.

Binding Sites↗
PubMed2003

Stable inheritance of telomere chromatin structure and function in the absence of telomeric repeats.

It is generally believed that telomeric repeats are a necessary and sufficient cis-element for telomere function. Here we show that telomere structure and meiotic function are stably inherited in fission yeast circular chromosomes that have lost all telomeric repeats. We found that the telomeric repeat binding protein, Taz1, and the heterochromatin protein, Swi6, remain associated with subtelomeres in the absence of telomeric repeats. We also found that the fusion point of circular chromosomes that lack telomeric repeats associates with SPB (the yeast counterpart of the centrosome) in the premeiotic horsetail stage, similarly to wild-type telomeres. However, a taz1+ deletion/reintroduction experiment revealed that the maintenance of Taz1 binding and premeiotic function is achieved via different strategies. Taz1 is recruited to subtelomeres by an autonomous element present in subtelomeric DNA, thus in a genetic mechanism. In contrast, the premeiotic subtelomere-SPB association is maintained in an epigenetic manner. These results shed light on the previously unrecognized role played by the subtelomere and underscore the robust nature of the functional telomere complex that is maintained by both genetic and epigenetic mechanisms. Furthermore, we suggest that the establishment and the maintenance of the functional telomere complex are mechanistically distinguishable.

Chromatin↗
PubMed2003

The fission yeast spSet1p is a histone H3-K4 methyltransferase that functions in telomere maintenance and DNA repair in an ATM kinase Rad3-dependent pathway.

We have characterized spSet1p, the Schizosaccharomyces pombe ortholog of the budding yeast histone H3 methyltransferase Set1p. SpSet1p catalyzes methylation of H3 at K4, in vivo and in vitro. Deleting spset1 partially affects telomeric and centromeric silencing. Strikingly, lack of spSet1p causes elongation of telomeres in wild-type cells and in most DNA damage checkpoint rad mutant cells, but not in cells lacking the ATM kinase Rad3 or its associated protein Rad26. Interestingly, spset1 deletion specifically causes a reduction in sensitivity to ultraviolet radiation of the PCNA-like checkpoint mutants hus1 and rad1, but not of cells devoid of Rad3. This partial suppression was not due to restoration of checkpoint function or to transcriptional induction of DNA repair genes. Moreover, spset1 allows recovery specifically of the crb2 checkpoint mutant upon treatment with the replication inhibitor hydroxyurea but not upon UV irradiation. Nevertheless, the pathway induced in spset1 cells cannot substitute for the Mus81/Rqh1 DNA damage tolerance pathway. Our results suggest that SpSet1p and the ATM kinase Rad3 function in a common genetic pathway linking chromatin to telomere length regulation and DNA repair.

Antineoplastic Agents↗