FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023990944> ?p ?o ?g. }

• W2023990944 endingPage "3853" @default.
• W2023990944 startingPage "3844" @default.
• W2023990944 abstract "Article9 September 2004free access Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance Blerta Xhemalce Corresponding Author Blerta Xhemalce Unité de la Dynamique du Génome, Institut Pasteur, Paris Cedex, France Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Jacob-S Seeler Jacob-S Seeler Unité Organisation Nucléaire et Oncogénèse, Institut Pasteur, Paris Cedex, France Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Geneviève Thon Geneviève Thon Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Anne Dejean Anne Dejean Unité Organisation Nucléaire et Oncogénèse, Institut Pasteur, Paris Cedex, France Search for more papers by this author Benoît Arcangioli Corresponding Author Benoît Arcangioli Unité de la Dynamique du Génome, Institut Pasteur, Paris Cedex, France Search for more papers by this author Blerta Xhemalce Corresponding Author Blerta Xhemalce Unité de la Dynamique du Génome, Institut Pasteur, Paris Cedex, France Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Jacob-S Seeler Jacob-S Seeler Unité Organisation Nucléaire et Oncogénèse, Institut Pasteur, Paris Cedex, France Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Geneviève Thon Geneviève Thon Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark Search for more papers by this author Anne Dejean Anne Dejean Unité Organisation Nucléaire et Oncogénèse, Institut Pasteur, Paris Cedex, France Search for more papers by this author Benoît Arcangioli Corresponding Author Benoît Arcangioli Unité de la Dynamique du Génome, Institut Pasteur, Paris Cedex, France Search for more papers by this author Author Information Blerta Xhemalce 1,3, Jacob-S Seeler2,3, Geneviève Thon3, Anne Dejean2 and Benoît Arcangioli 1 1Unité de la Dynamique du Génome, Institut Pasteur, Paris Cedex, France 2Unité Organisation Nucléaire et Oncogénèse, Institut Pasteur, Paris Cedex, France 3Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen K, Denmark ‡These authors contributed equally to this work *Corresponding authors. Unité de la Dynamique du Génome, URA1664 du CNRS, Jacques Monod Building, Institut Pasteur, 25, rue du Dr Roux, 75724, Paris Cedex 15, France. Tel.: +33 1 4568 8454; Fax: +33 1 4568 8960; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2004)23:3844-3853https://doi.org/10.1038/sj.emboj.7600394 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sumoylation represents a conserved mechanism of post-translational protein modification. We report that Pli1p, the unique fission yeast member of the SP-RING family, is a SUMO E3 ligase in vivo and in vitro. pli1Δ cells display no obvious mitotic growth defects, but are sensitive to the microtubule-destabilizing drug TBZ and exhibit enhanced minichromosome loss. The weakened centromeric function of pli1Δ cells may be related to the defective heterochromatin structure at the central core, as shown by the reduced silencing of an ura4 variegation reporter gene inserted at cnt and imr. Interestingly, pli1Δ cells also exhibit enhanced loss of the ura4 reporter at these loci, likely by gene conversion using homologous sequences as information donors. Moreover, pli1Δ cells exhibit consistent telomere length increase, possibly achieved by a similar process. Point mutations within the RING finger of Pli1p totally or partially reproduce the pli1 deletion phenotypes, thus correlating with their sumoylation activity. Altogether, these results strongly suggest that Pli1p, and by extension sumoylation, is involved in mechanisms that regulate recombination in particular heterochromatic repeated sequences. Introduction Covalent protein modification by SUMO (sumoylation) has been shown to play important roles in such diverse processes as nucleo-cytoplasmic transport, chromosome segregation, DNA metabolism, as well as transcriptional regulation (reviewed in Melchior, 2000; Müller et al, 2001; Seeler and Dejean, 2003). Like the modification with ubiquitin and other ubiquitin-like modifiers (Ubls), sumoylation is achieved with a distinct but evolutionarily conserved pathway consisting of E1-activating, E2-conjugating and E3 ligase enzymes. Sumoylation of numerous target proteins can be achieved in vitro using only E1 and E2 enzymes; however, in vivo, it is likely that the substrate specificity and the rate of the reaction depend critically on the activity of E3 ligase-containing complexes, as is also the case for ubiquitylation. To date, three protein families have been implicated as SUMO E3 ligases. The first, or SIZ/protein inhibitor of activated Stat (PIAS) family, was initially shown in budding yeast (Saccharomyces cerevisiae) to mediate the modification of septins (Johnson and Gupta, 2001; Takahashi et al, 2001). Subsequently, members of the mammalian PIAS family were shown to be critical for the modification of transcription factors such as p53, nuclear receptors (e.g. AR, PR), Lef1 and Sp3 (Jackson, 2001; Kotaja et al, 2002; Sapetschnig et al, 2002; Gill, 2003). A second type of E3 ligase is given by the nuclear import factor RanBP2, which has been shown to mediate the modification of the nuclear proteins SP100 and HDAC4 (Kirsh et al, 2002; Pichler et al, 2002), and has provided a further link between sumoylation and nucleo-cytoplasmic transport. Finally, a third SUMO E3 ligase was discovered with the polycomb protein Pc2, which was found to enhance the modification of the transcriptional repressor CtBP1 (Kagey et al, 2003). Studies of sumoylation in mammalian cells have largely been focused on the effects on transcriptional regulation, largely because the majority of the currently characterized SUMO substrates are either transcription factors or co-factors. A growing number of cases supports a model in which the sumoylation of a transcription factor leads to repression (reviewed in Verger et al, 2003). Consistent with this is the finding that the sumoylation target sites often fall within known repression domains of dual-function transcription factors and that mutation of these sites leads to de-repression. While the precise mechanisms involved remain elusive, it has been shown in some cases (Lef1, Sp3) that sumoylation leads to the sequestration of the transcription factor to specific nuclear domains, such as PML nuclear bodies, and thus to the concomitant attenuation of transcriptional activity. That sumoylation may also play a role at the DNA (or promoter) level has been suggested by Shiio and Eisenman (2003), who showed that the E2 enzyme (Ubc9) fused to a GAL4 DNA-binding domain could lead to transcriptional repression of a reporter gene that is associated with the appearance of sumoylated chromatin, presumably at histone H4 within nucleosomes. Analysis of the role of sumoylation in genetically tractable organisms has opened new fields of interest such as chromosome condensation and cohesion, and DNA replication and repair. In the budding yeast, the proliferating cell nuclear antigen (PCNA) was shown to be modified by ubiquitin and SUMO on the same lysine residue (K164), sumoylation occurring during S phase and ubiquitylation upon DNA damage, channeling DNA repair towards the RAD6-dependent post-replication repair mechanisms (Hoege et al, 2002; Stelter and Ulrich, 2003). It was proposed that sumoylation of PCNA has an inhibitory function on repair since abrogation of SUMO modification by mutating both K164 and K127 (the other residue subject to sumoylation) partially rescues the DNA damage sensitivity of the PCNA K164R single mutant. Again, in budding yeast, SMT4, a de-sumoylation enzyme, was found as a high copy suppressor of thermosensitive alleles of the SMC2 gene, encoding one of the core condensin subunits (Strunnikov et al, 2001) and of the PDS5 gene, required for proper chromatid cohesion maintenance (Stead et al, 2003). Furthermore, SMT4 mutants exhibit severe defects in chromatin condensation (Strunnikov et al, 2001) and precocious dissociation of sister chromatids during mitosis, which has been linked to the sumoylation of two major proteins TOP2 and PDS5 (Bachant et al, 2002; Stead et al, 2003). In Drosophila melanogaster, the Su(var)2–10 gene, which encodes a member of the Siz/PIAS family, is required for viability, and mutants show defects in both chromosome condensation and inheritance (Hari et al, 2001). In Schizosaccharomyces pombe, mutations in the genes encoding SUMO (Pmt3p; Tanaka et al, 1999), E1 (Rad31p; Shayeghi et al, 1997), E2 (Hus5p; al-Khodairy et al, 1995) in the SUMO pathway produce cells that survive but display severe growth defects, that is, are essentially nonviable, and thus preclude proper genetic analysis. In this work, we report the characterization of a novel fission yeast nuclear protein, Pli1p, as an E3 ligase in the SUMO pathway. Deletion of the pli1 gene, while exhibiting only mild or no mitotic phenotypes, leads to increased sensitivity to the microtubule spindle poison TBZ and minichromosome loss, indicating improper centromere and/or kinetochore function. Consistent with this, pli1Δ cells display alleviated silencing of an ura4 reporter gene at the central centromeric core required for kinetochore assembly. Furthermore, we present evidence for a role of Pli1p in protecting heterochromatic repeated sequences (i.e. centromeres and telomeres) from illegitimate recombination. The phenotypes described for pli1-deleted cells are totally or partially reproduced by point mutations within the RING finger, in correlation with their E3 ligase activity, thus strongly suggesting that sumoylation via Pli1p is involved in centromeric and telomeric functions in fission yeast. Results Isolation of Pli1p, an S. pombe Siz1/Siz2/PIAS homologue Our previous studies of the biological role of the essential chromatin-associated protein switch-activating protein 1 (Sap1p) (De Lahondes et al, 2003) prompted us to perform a yeast two-hybrid screen of a S. pombe cDNA library with this protein as bait. While the functional relevance of the obtained interactants remains to be elucidated, this screen identified a novel cDNA corresponding to the hypothetical open reading frame (ORF) listed as SPAC1687.05 under the reserved name of pli1 in the Sanger Institute GeneDB database. The pli1+ ORF translates into a putative protein of 727 amino acids with a predicted molecular mass of 80.7 kDa. Amino-acid sequence comparisons revealed significant similarity to the SIZ1 and SIZ2 proteins of budding yeast as well as all known mammalian PIAS proteins. Pli1p notably shares with these two well-conserved domains: an amino-terminal SAP (SAF-A/B, Acinus and PIAS) domain and a central SP-RING (Siz/PIAS-RING) domain (Hochstrasser, 2001), essential for the SUMO E3 ligase activity associated with the Siz/PIAS proteins (Seeler and Dejean, 2003) (Figure 1). Western blot analysis of WCE of cells containing an endogenous HA (data not shown) or CFP-tagged version of the protein (Figure 2B) confirmed the existence and size of the Pli1p protein. Further immunofluorescence analysis using an anti-GFP antibody to detect the endogenous Pli1-CFP protein revealed that Pli1p is localized in the nucleus, where it forms numerous spots (Figure 2A). Figure 1.Sequence alignment of the SAP and SP-RING domains of Pli1p and several (putative) SP-RING SUMO ligases. Alignment of S. pombe Pli1p, S. cerevisiae Siz2p, Siz1p, D. melanogaster Zimp-B and Homo sapiens hPIASy protein sequences (GenBank accession numbers CAA22599.1, NP_014799, NP_010697, AAD29288, Q8N2W9, respectively), using CLUSTAL X algorithm. Identical residues are boxed and similar residues are shaded. Overall sequence identity of Pli1p with the other four orthologs is 26–27% (33–38% similarity), whereas the SAP domain has 22–35% identical residues (75–68% similarity) and the SP-RING domain 39–51% (70–75% similarity). Asterisks indicate the positions of the cysteiyl and histidyl residues forming the C2HC3 conserved SP-RING domain. Download figure Download PowerPoint Figure 2.Pli1p is localized in the nucleus and forms numerous foci. (A) WT (PB46) and pli1-CFP (BX4) cells grown exponentially to 5 × 106 cells in MM at 33°C were fixed with paraformaldehyde and stained with anti-CFP rabbit polyclonal antibody, followed by FITC-conjugated anti-rabbit IgG secondary antibody and DAPI. Z-scanning of cells every 0.4 μm showed that the CFP-specific spots observed in pli1-CFP cells were present throughout the nucleus. (B) Anti-CFP Western blot of denaturing protein whole-cell extracts of WT (PB46) and pli1-CFP (BX4) cells grown in the same conditions as above. Download figure Download PowerPoint Pli1p promotes SUMO/Pmt3p conjugation in vivo and in vitro The homology of Pli1p to the SIZ1/2 budding yeast and mammalian PIAS proteins suggests that Pli1p might act as a SUMO E3 ligase. We therefore first analyzed the effect of pli1 deletion on the in vivo pattern of SUMO conjugates (Figure 3A) Wild-type and pli1Δ cells containing or not an N-terminal His6 tag fused in frame with the endogenous pmt3 gene were analyzed by Western blotting using a rabbit antibody raised against the S. pombe SUMO/Pmt3p protein. pli1Δ extracts showed an increase in the intensity of free Pmt3p and an overall severe decrease in the intensity of Pmt3p-specific bands corresponding to Pmt3p conjugates, indicating a significant reduction of global sumoylation in pli1-deleted cells. Figure 3.Pli1p promotes SUMO conjugation in vivo and in vitro. (A) Whole-cell extracts of WT, pli1C321S and pli1Δ cells were separated on denaturing 8% (top panel) and 15% (middle panel) SDS–polyacrylamide gels and Western blotted with anti-Pmt3p antiserum. The specificity of bands revealed by the anti-Pmt3p polyclonal antibody is shown by their slight upward shift in the His6-pmt3 strain carrying the endogenous pmt3 gene fused in frame with an amino-terminal His6 tag. The arrows indicate unconjugated Pmt3p; asterisks indicate crossreacting bands. Strains used: PB46 (WT); BX6 (pli1C321S); BX2 (Δpli1); BX5 (his6-pmt3). (B) Sumoylation reactions with in vitro-translated, 35S-labeled Rad22p substrate (4 μl per reaction in 22 μl total) and recombinant, bacterially produced Pmt3p (Pmt3-GG, 1 μg in lanes 2–7), E1 (GST–Rad31p–Fub2p, 32 ng), E2 (Hus5p, 80 ng in lanes 1–2 and 4–7; 400 ng in lane 3) and WT or Pli1C321S (4.8; 24; 120; 600 ng in lanes 4–7, respectively) proteins. The fastest migrating band of the modified species (i.e. mono-modified Rad22) was quantitated by PhosphorImager analysis and expressed as a percentage of total Rad22p. (C) Reaction time course of Rad22p in vitro modification. Standard reactions (4 μl 35S-Rad22p, 1 μg Pmt3-GG, 32 ng E1, 80 ng E2) were assembled, containing 8 ng of WT Pli1p (open squares), Pli1pC321S (open triangles) or Pli1pC321S/H323A/C326S (open circles), and incubated for the indicated times. Open and closed diamonds represent modification without added E3 at 80 and 400 ng of E2 (Hus5p), respectively. Percent total modification was quantitated by PhosphorImager analysis. Download figure Download PowerPoint To demonstrate that Pli1p possesses SUMO E3 ligase activity, we employed a modification system consisting of recombinant Pmt3p and the E1 (Rad31p-Fub2p) and E2 (Hus5p) enzymes to modify Rad22p, a previously described S. pombe SUMO substrate (Ho et al, 2001). As shown in Figure 3B, 35S-labelled, in vitro translated Rad22p is efficiently modified in the absence of Pli1p at high concentrations of E2 enzyme (lane 2). At one-fifth the concentration of E2, Rad22p modification was limited (lane 3) but could be restored by the addition of recombinant Pli1p (lanes 4–7) in a dose-dependent manner. In vitro binding assays showed that Pli1p interacts with both the E2 enzyme (Hus5p), as well as with the Rad22p substrate (data not shown), thus demonstrating that Pli1p fulfills the criteria for SUMO E3 ligase function. Furthermore, by introducing point mutations of either C321S or C321S/H323A/C326S within the SP-RING finger (Figure 1), we obtained Pli1 proteins which partially or totally lost the capacity to enhance the modification of Rad22p in the in vitro sumoylation system (Figure 3B and C). Indeed, addition of the inactive Pli1p C321S/H323A/C326S mutant to the reaction reduced sumoylation to levels somewhat below those obtained in its absence, possibly because Pli1p itself is a SUMO substrate in vitro (data not shown) and, when inactive, behaves as a competitor for sumoylation. When introduced in S. pombe cells, the pli1C321S WCE showed intermediate levels of global sumoylation between WT and pli1Δ cells (Figure 3A), whereas the C321S/H323A/C326S mutant behaved like the pli1Δ cells (data not shown). pli1 is involved in centromeric function In S. pombe, the pmt3 null mutant displays severe morphological abnormalities and hypersensitivity to various stress conditions (Tanaka et al, 1999), while the hus5 (encoding Ubc9p) null allele is almost inviable (al-Khodairy et al, 1995). In contrast, pli1-deleted strain grew well and lacked hypersensisitivity to DNA-damaging agents like UV, MMS and hydroxy-urea (data not shown). However, pli1Δ cells were sensitive to the microtubule-destabilizing drug thiabendazole (TBZ) (Figure 4A). As TBZ sensitivity is frequently associated with defects in centromeric function, we then tested the chromosome stability in pli1Δ cells, which indeed showed a 10-fold increase in the frequency of the Ch16 minichromosome loss (Figure 4B). We further analyzed the effect of pli1 deletion on the heterochromatic function of S. pombe centromeres. In fission yeast, the centromeres are large DNA structures (40–100 kb) composed of outer repetitive regions (otr) corresponding to the pericentromeric heterochromatin and central regions (innermost repeat imr and central cnt), which form specialized SpCENP-A-dependent chromatin, and previous studies have shown that transcription of genes inserted into these domains is silenced (Chikashige et al, 1989; Niwa et al, 1989; Clarke and Baum, 1990; Allshire et al, 1994, 1995; Baum et al, 1994; Ekwall et al, 1999; Partridge et al, 2000). We assayed silencing of the ura4+ gene inserted at three different loci in CEN1 (Centromere of Chromosome I): the center region (cnt1∷ura4+), the innermost repeat (imr∷ura4+) and the outer repeat (otr∷ura4+) (Figure 4C) in WT and pli1Δ strains, by the ability of cells to survive in the presence of 5-fluoro-orotic acid (FOA), which generates a toxic metabolite in (ura+) strains. In WT cells, the inserted ura4+ variegates between expressed and repressed states as cells can grow both on −URA and FOA plates. However, deletion of pli1 causes a significant decrease in silencing at the imr1∷ura4+ and, in a milder way, at the somewhat more expressed cnt1∷ura4+ insertion sites, as indicated by the lower efficiency of plating on FOA plates of these cells (Figure 4D). These results were also confirmed by Northern blot analysis using an ura4 probe (data not shown). The use of the pli1C321S and pli1C321S/H323A/C326S mutants in these experiments showed a strong correlation between SUMO E3 ligase activity and the centromeric phenotypes, since the pli1C321S single mutant showed intermediate levels of TBZ sensitivity, minichromosome loss and silencing at the central region between WT and pli1Δ cells (Figure 4) and the triple mutant exactly reproduced the pli1 deletion phenotypes (data not shown). Figure 4.Effects of pli1 mutants on centromeric function. (A) pli1 cells are sensitive to the microtubule-destabilizing drug thiabendazole (TBZ): Serial five-fold dilutions of exponential cultures of WT (PB10), pli1C321S (BX7) and pli1Δ (BX1) cells were spotted to YES and YES plates containing TBZ (17.5 or 20 μg/ml) and incubated for 4 days at 33°C. (B) pli1 cells show an increased frequency of minichromosome loss. Independent colonies of WT (JS161), pli1 C321S (BX9) and pli1Δ (BX8) strains grown in adenine selective media, to select for the presence of the Ch16 minichromosome, were plated to YE plates at a concentration of 103 cells/plate and incubated for 4 days at 33°C. The values shown are the average of the percentage of red colonies calculated for 16 independent colonies in two independent experiments. (C, D) pli1 deletion affects silencing at the central core of centromeres. Expression of the ura4+ gene inserted at three different positions of centromere 1, schematically represented in (C), was assayed by plating efficiency of serial three-fold (cnt) or five-fold (imr and otr) dilutions of cell suspensions spotted on selective media shown in (D). Strains used: cnt1TM1(NcoI)∷ura4+: WT=FY336, pli1C321S=BX10, pli1Δ=PG2964; imr1R(NcoI)∷ura4+: WT=FY498, pli1C321S=BX12, pli1Δ=PG2972; otr1R(SphI)∷ura4+: WT=FY648, pli1C321S=BX14, pli1Δ=PG2976. Download figure Download PowerPoint Implication of pli1 in the sequence stability of centromeres While assaying silencing of the ura4+ gene inserted at cnt1 and imr1R, we noted that pli1 mutant cells recurrently gave rise to large colonies on FOA plates (Figure 4D) that turned out to be unable to grow on −URA plates. As this does not correspond to a variegation phenotype (Allshire et al, 1994), we analyzed them at the DNA level. The central domain of CEN1 is composed of two 5.6 kb inverted repeats (imr1R and imr1L) surrounding a 4.1 kb center (cnt1), which otherwise partially shares its sequence with the central domain of CEN 3 (Takahashi et al, 1992) (Figure 5A). The ura4 gene was inserted at the NcoI sites of cnt1 and imr1R (Allshire et al, 1994, 1995). Challenging independent FOA papillae from WT and pli1Δ strains carrying the ura4+ gene at cnt1 and imr1R for growth on −URA plates indicated that most, if not all, papillae from pli1 mutants were stable (ura4−), whereas papillae from WT could grow both on FOA and −URA plates as expected (Allshire et al, 1994). Genomic DNA from 20 independent (ura−) clones of the pli1Δ, CEN1-imr1R(NcoI)∷ura4+ (PG2972) strain was digested with KpnI and analyzed by a Southern blot sequentially using a cnt1 and an ura4 probe (Figure 5B). This analysis revealed that all isolated clones had deleted the ura4 gene at imr1R. Further Southern blot analysis using an NcoI digestion and an imr1 probe revealed that the deletion of the ura4 gene had restored the WT sequence, as indicated by the presence of an NcoI site at imrR1 (Figure 5B). Since imr1R and imr1L are identical over 99% of their sequence, the simplest interpretation would be that the ura4 gene inserted at imr1R was deleted following gene conversion events using the homologous sequence at the inverted repeat imr1L as template. A similar result was obtained with 19 over 20 independent (ura−) clones of the pli1Δ, CEN1-cnt1(NcoI)∷ura4+ (PG2964) strain (see Supplementary Figure S1). Since cnt1 also shares 100% identity over 3.3 kb with the central domain of CEN3, these latter clones likely arose from interchromosomal gene conversion events. The frequency of ura4 loss was evaluated at ≈10−3 for both domains in pli1Δ cells and, as stable (ura−) cells could not be isolated from 104 wild type cells, ura4 deletion is therefore increased by at least 10-fold in pli1Δ compared to WT cells. This effect seems to be specific for the centromeric loci, since an artificial intrachromosomal recombination reporter (van den Bosch et al, 2002) at a euchromatic locus displayed no difference between pli1Δ and WT cells (see Supplementary Figure S2). Figure 5.Effect of pli1 deletion on the stability of ura4+ gene inserted at imr1R. (A) Structure of centromere 1 central domain in CEN1+ and CEN1: imr1R(NcoI)∷ura4+ strains. KpnI and NcoI sites, sizes of their respective digestion fragments and positions of the probes used for the Southern blot analysis are indicated. Note that the cnt1 and ura4 probes also recognize homologous sequences, respectively, at CEN3 and ura4D/SE loci, thus serving as internal controls. (B) Southern blot analysis of stable [ura−] independent clones raising from CEN1-imr1R(NcoI)∷ura4 deleted for pli1 gene. After digestion with KpnI, genomic DNA prepared in agarose plugs was subjected to pulsed field electrophoresis on 1.2% agarose gel and analyzed by a Southern Blot using the cnt1 probe. The membrane was then stripped and reprobed with the ura4 probe. Genomic DNA was also digested with NcoI, subjected to electrophoresis on 0.75% agarose gel, transferred to a nylon membrane and hybridized to the imr1 probe. Sizes or identities of bands are indicated. Strains used: CEN1+: PB10; CEN1-imr1R(NcoI)∷ura4+: WT=FY498, pli1Δ=PG2972; and 20 [ura−] independent clones isolated from PG2972 strain=BX13_1–20. Download figure Download PowerPoint Pli1p is involved in the control of telomere length While examining silencing at three other heterochromatic loci: the mat2/3 inactive sexual locus, the rDNA and the telomeres, we noticed that pli1 mutants slightly increased the silencing of the ura4+ gene next to the telomeric repeat of the Ch16m23∷ura4+ TEL[72] minichromosome without affecting silencing at the two other loci (data not shown). As the frequency of transcriptional repression at the telomere can be increased by longer telomeres (Kyrion et al, 1993) and, further, since deletion of pmt3 was previously reported to lead to telomere elongation (Tanaka et al, 1999), we assessed telomere length in pli1 cells. Genomic DNA digested with EcoRI was analyzed by a Southern blot using a 32P end-labeled telomeric oligonucleotide (Cooper et al, 1997) (Figure 6B). The EcoRI site is located ≈0.8 kb away from the telomeric repeats sequences, giving rise to a broad telomere hybridization signal centered at 950 bp in the WT strain (Figure 6A, lane 1). The pli1Δ strain showed a clearly upshifted, more intense signal that ranges from 1 to 1.4 kb (lane 3), corresponding to a telomere length intermediate between that of WT and pmt3Δ cells (Tanaka et al, 1999). The pli1C321S single mutant again showed a milder effect on the increase of telomere length (lane 2), while the pli1C321S/H323A/C326S triple mutant showed the same effect as the pli1 deletion (data not shown). Figure 6.Genetic interactions of pli1 and recombination genes: effects on telomere length. (A) Genetic interaction of pli1 with homologous recombination mutants. Three representative tetrads from diploids heterozygous for disruption of pli1 and either rad50 (BX16), rad22 (BX18) or rad51 (BX19), and three representative tetrads from the rad51(JAC1/51Δ) × pli1C321S (BX7) cross, as indicated. The genotype of each segregant was determined (or inferred) by replica plating on appropriate selective media and/or by PCR; double-mutant clones are indicated by diamonds. Given the close physical linkage of the rad22 and pli1 loci (0.05 Morgans), tetrad analysis of the rad22+/− pli1+/− diploïd (250 tetrads) was complemented by extensive random spore analysis (data not shown). (B) Southern blot analysis of telomere length in (1) WT (PB10); (2) pli1C321S (BX7); (3) pli1Δ (BX1); (4) pli1Δ/rad50Δ (BX20); (5) rad50Δ (BX21); (6) pli1C321S/rad51Δ (BX22_1); (7) rad51Δ (BX22_2); (8) pli1C321SΔ (BX22_3); (9) WT (BX22_4). BX22_1–4 correspond to the four clones of a tetra-type tetrad from the rad51(JAC1/51Δ) × pli1C321S (BX7) cross. After digestion with EcoRI, genomic DNA was subjected to electrophoresis on 1% agarose gel, transferred to a nylon membrane, and hybridized to a telomeric DNA probe (Cooper et al, 1997). Download figure Download PowerPoint To test whether the increase in telomere length in pli1 mutants could have resulted from normally repressed gene conversion events, we performed a genetic analysis involving pli1 and three major genes involved in homologous recombination: rad22 (fission yeast RAD52 homolog), rad50 and rad51/rhp51. We therefore produced diploids doubly heterozygous for pli1 and each of the three rad genes by sequential disruption. At least 100 tetrads from microdissected asci were genotyped for each experiment and representative examples are shown in Figure 6A. The pli1-rad50 double mutants showed a reduced viability (50%) and a significant increase in generation time independent of mating type (data not shown). They also exhibited a significant increase in telomere length compared to the rad50Δ single mutant (Figure 6B, compare lanes 4 and 5), but very similar to that observed in pli1Δ cells (lane 3). In contrast, disruptants of pli1 and either rad22 or rad51 displayed synthetic lethality, in that double-mutant clones gave rise to microcolonies of 8–150 cells that contained numerous abnormally elongated cells (data not shown). In order to possibly circumvent the lethality of pli1–rad51 double mutants, we crossed the pli1C321S and rad51 mutants and found that the obtained pli1C321S–rad51Δ double mutants were viable, although they grew slower than the rad51 single mutants of the same mating type (Figure 6A). When we next analyzed clones from tetra-type tetrads of the pli1C321S × rad51Δ cross at the telomere level (Figure 6B, lanes 6–9), we found that pli1C321S–rad51Δ clones (lane 6) showed a telomere length similar to the rad51Δ single mutant (lane 7" @default.
• W2023990944 created "2016-06-24" @default.
• W2023990944 creator A5002003012 @default.
• W2023990944 creator A5027055248 @default.
• W2023990944 creator A5064665613 @default.
• W2023990944 creator A5077327911 @default.
• W2023990944 creator A5077482316 @default.
• W2023990944 date "2004-09-09" @default.
• W2023990944 modified "2023-10-11" @default.
• W2023990944 title "Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance" @default.
• W2023990944 cites W165187905 @default.
• W2023990944 cites W1744223828 @default.
• W2023990944 cites W1837587701 @default.
• W2023990944 cites W1864629140 @default.
• W2023990944 cites W1875299626 @default.
• W2023990944 cites W1891192204 @default.
• W2023990944 cites W195095803 @default.
• W2023990944 cites W1951655399 @default.
• W2023990944 cites W1971503458 @default.
• W2023990944 cites W1974875206 @default.
• W2023990944 cites W1978143896 @default.
• W2023990944 cites W1978197643 @default.
• W2023990944 cites W1979524912 @default.
• W2023990944 cites W1982758939 @default.
• W2023990944 cites W1991326375 @default.
• W2023990944 cites W2001029086 @default.
• W2023990944 cites W2006247396 @default.
• W2023990944 cites W2007452601 @default.
• W2023990944 cites W2026111974 @default.
• W2023990944 cites W2029234227 @default.
• W2023990944 cites W2032360849 @default.
• W2023990944 cites W2040825545 @default.
• W2023990944 cites W2042377388 @default.
• W2023990944 cites W2056526146 @default.
• W2023990944 cites W2060817283 @default.
• W2023990944 cites W2070163476 @default.
• W2023990944 cites W2078362264 @default.
• W2023990944 cites W2083312621 @default.
• W2023990944 cites W2086438431 @default.
• W2023990944 cites W2094801025 @default.
• W2023990944 cites W2095191110 @default.
• W2023990944 cites W2097160587 @default.
• W2023990944 cites W2112575409 @default.
• W2023990944 cites W2119301405 @default.
• W2023990944 cites W212138329 @default.
• W2023990944 cites W2121428233 @default.
• W2023990944 cites W2121779606 @default.
• W2023990944 cites W2122044343 @default.
• W2023990944 cites W2123994351 @default.
• W2023990944 cites W2130303330 @default.
• W2023990944 cites W2136053271 @default.
• W2023990944 cites W2139114666 @default.
• W2023990944 cites W2140138777 @default.
• W2023990944 cites W2140321354 @default.
• W2023990944 cites W2140513365 @default.
• W2023990944 cites W2141455492 @default.
• W2023990944 cites W2141764062 @default.
• W2023990944 cites W2143883087 @default.
• W2023990944 cites W2155488858 @default.
• W2023990944 cites W2165427373 @default.
• W2023990944 cites W2168562183 @default.
• W2023990944 cites W2175924795 @default.
• W2023990944 cites W2194986544 @default.
• W2023990944 cites W2791163698 @default.
• W2023990944 cites W861725403 @default.
• W2023990944 doi "https://doi.org/10.1038/sj.emboj.7600394" @default.
• W2023990944 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/522793" @default.
• W2023990944 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15359282" @default.
• W2023990944 hasPublicationYear "2004" @default.
• W2023990944 type Work @default.
• W2023990944 sameAs 2023990944 @default.
• W2023990944 citedByCount "68" @default.
• W2023990944 countsByYear W20239909442012 @default.
• W2023990944 countsByYear W20239909442013 @default.
• W2023990944 countsByYear W20239909442014 @default.
• W2023990944 countsByYear W20239909442015 @default.
• W2023990944 countsByYear W20239909442017 @default.
• W2023990944 countsByYear W20239909442018 @default.
• W2023990944 countsByYear W20239909442019 @default.
• W2023990944 countsByYear W20239909442022 @default.
• W2023990944 crossrefType "journal-article" @default.
• W2023990944 hasAuthorship W2023990944A5002003012 @default.
• W2023990944 hasAuthorship W2023990944A5027055248 @default.
• W2023990944 hasAuthorship W2023990944A5064665613 @default.
• W2023990944 hasAuthorship W2023990944A5077327911 @default.
• W2023990944 hasAuthorship W2023990944A5077482316 @default.
• W2023990944 hasBestOaLocation W20239909441 @default.
• W2023990944 hasConcept C104317684 @default.
• W2023990944 hasConcept C121332964 @default.
• W2023990944 hasConcept C12294094 @default.
• W2023990944 hasConcept C134459356 @default.
• W2023990944 hasConcept C152568617 @default.
• W2023990944 hasConcept C175114707 @default.
• W2023990944 hasConcept C177336024 @default.
• W2023990944 hasConcept C25602115 @default.
• W2023990944 hasConcept C2776519988 @default.
• W2023990944 hasConcept C2777576037 @default.
• W2023990944 hasConcept C2779222958 @default.

• next