Evidence for a Role of MCM (Mini-chromosome Maintenance)5 in Transcriptional Repression of Sub-telomeric and Ty-proximal Genes in Saccharomyces cerevisiae
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m301110200
ISSN1083-351X
AutoresRenata Dziak, David Leishman, Maja Radovic, Bik K. Tye, Krassimir Yankulov,
Tópico(s)RNA Research and Splicing
ResumoThe MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons. The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons. MCM 1The abbreviations used are: MCM, mini-chromosome maintenance; ORC, origin recognition complex; CDC, cell division cycle; LTR, long terminal repeat; TPE, telomere position effect; SIR, silencing information region; FOA, fluorotic acid; FOAR, FOA-resistant; COS, conserved sequences at telomeres; ORF, open reading frame; HM, mating type loci; pol, polymerase; STAT1, signal transducers and activators of transcription 1; ARS, autonomously replicating sequence; SAGA/ADA, SPT-ADA-GCN5 acetylase ADAptor. (mini-chromosome maintenance) genes had been identified in screens for Saccharomyces cerevisiae mutants, which displayed high rates of minichromosome loss (1Tye B.K. Methods. 1999; 18: 329-334Crossref PubMed Scopus (33) Google Scholar). MCM2-MCM7 homologues were subsequently found in all eukaryotes. It is believed that MCM proteins act as a component of the licensing machinery, which limits the occurrence of DNA replication to once per cell cycle (2Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). MCM proteins form pre-replicative complexes at active as well as silent origins in S. cerevisiae or on un-replicated DNA in higher eukaryotes in a manner dependent on origin recognition complex (ORC) and Cdc6 (2Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). Activation of these pre-replicative complexes by protein kinases is required for initiation of replication (2Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). Removal of the MCM complex from origins at the time of initiation is believed to limit origin firing until the next cell cycle when the pre-replicative complex is re-established (2Lei M. Tye B.K. J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 3Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). MCM proteins also seem to have a post-initiation role in DNA replication as indicated by their association with moving replication forks (4Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar) and by the requirement for their uninterrupted function for fork progression in S. cerevisiae (5Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (523) Google Scholar). In vitro assembled MCM complexes have also been shown to have DNA helicase activity consistent with a role in both melting of origin DNA and unwinding during fork elongation (3Labib K. Diffley J.F. Curr. Opin. Genet. Dev. 2001; 11: 64-70Crossref PubMed Scopus (125) Google Scholar). Some of the properties of MCM proteins suggest that they may have functions that extend beyond the regulation of DNA replication. They are 100- to 1000-fold more abundant than the estimated number of origins in S. cerevisiae (6Lei M. Kawasaki Y. Tye B.K. Mol. Cell. Biol. 1996; 16: 5081-5090Crossref PubMed Scopus (155) Google Scholar, 7Young M.R. Tye B.K. Mol. Biol. Cell. 1997; 8: 1587-1601Crossref PubMed Scopus (68) Google Scholar) and mammals (8Burkhart R. Schulte D. Hu D. Musahl C. Goehring F. Knippers R. Eur. J. Biochem. 1995; 228: 431-438Crossref PubMed Scopus (119) Google Scholar, 9Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar). Mammalian MCMs have been reported to associate with large complexes containing or contacting RNA polymerase II and general pol II transcription factors (10Yankulov K. Todorov I. Romanowski P. Licatalosi D. Cilli K. McCracken S. Laskey R. Bentley D.L. Mol. Cell. Biol. 1999; 19: 6154-6163Crossref PubMed Scopus (74) Google Scholar, 11Holland L. Gauthier L. Bell-Rogers P. Yankulov K. Eur. J. Biochem. 2002; 269: 5192-5202Crossref PubMed Scopus (27) Google Scholar), with the MAT1 component of CAK/TFIIH (12Wang Y. Xu F. Hall F.L. FEBS Lett. 2000; 484: 17-21Crossref PubMed Scopus (11) Google Scholar), with the transcriptional activator STAT1 (13Zhang J.J. Zhao Y. Chait B.T. Lathem W.W. Ritzi M. Knippers R. Darnell Jr., J.E. EMBO J. 1998; 17: 6963-6971Crossref PubMed Scopus (191) Google Scholar, 14DaFonseca C.J. Shu F. Zhang J.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3034-3039Crossref PubMed Scopus (69) Google Scholar), and with the tumor suppressor pRB (15Sterner J.M. Dew-Knight S. Musahl C. Kornbluth S. Horowitz J.M. Mol. Cell. Biol. 1998; 18: 2748-2757Crossref PubMed Scopus (165) Google Scholar, 16Gladden A.B. Diehl J.A. J. Biol. Chem. 2003; 278: 9754-9760Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) thus suggesting a function of MCMs in pol II transcription and gene expression. Other studies have linked MCM proteins to chromatin remodeling based on the interaction between the MCM proteins and histone H3/H4 dimers (17Ishimi Y. Komamura Y. You Z. Kimura H. J. Biol. Chem. 1988; 273: 8369-8375Abstract Full Text Full Text PDF Scopus (115) Google Scholar, 18Ishimi Y. Ichinose S. Omori A. Sato K. Kimura H. J. Biol. Chem. 1996; 271: 24115-24122Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and between MCM2 and the histone acetyltransferase HBO1 (19Burke T.W. Cook J.G. Asano M. Nevins J.R. J. Biol. Chem. 2001; 276: 15397-15408Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Genome-wide analyses of initiation of DNA replication in S. cerevisiae (20Raghuraman M.K. Winzeler E.A. Collingwood D. Hunt S. Wodicka L. Conway A. Lockhart D.J. Davis R.W. Brewer B.J. Fangman W.L. Science. 2001; 294: 115-121Crossref PubMed Scopus (618) Google Scholar, 21Newlon C.S. Theis J.F. Curr. Opin. Genet. Dev. 1993; 3: 752-758Crossref PubMed Scopus (198) Google Scholar) mapped 332 origins of replication. However, genome-wide mapping of the association of ORC and MCM proteins to chromatin (22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar) indicated at least 429 binding sites for these complexes. Only 79% of the ORC·MCM binding sites on chromosome X serve as origins. Another notable feature of ORC·MCM distribution is the clustering of ORC·MCM at telomeres, near Ty-transposable elements as well as solo Ty-LTR elements. Although telomeric X and Y′ elements are known to contain inactive or late replicating ARS elements where pre-replicative complexes could assemble (23Chan C.S. Tye B.K. Cell. 1983; 33: 563-573Abstract Full Text PDF PubMed Scopus (196) Google Scholar), Ty and solo Ty-LTR elements are not known to associate with replication origins. It was suggested that this association of ORC·MCM with extensive repetitive elements could influence chromatin organization and genome stability (22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar). Gene expression in the proximity of telomeres is reversibly repressed to produce either actively transcribed or completely silent chromatin, a phenomenon referred to as TPE (telomere position effect) (24Tham W.H. Zakian V.A. Oncogene. 2002; 21: 512-521Crossref PubMed Google Scholar). Mutations in the major regulators of TPE, the SIR genes and RAP1, also have an effect on the silent mating type loci (HM) and on rRNA gene repression (25Aparicio O.M. Billington B.L. Gottschling D.E. Cell. 1991; 66: 1279-1287Abstract Full Text PDF PubMed Scopus (608) Google Scholar, 26Roy N. Runge K.W. Curr. Biol. 2000; 10: 111-114Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). On the other hand, HDF1 and HDF2 are required for silencing at telomeres, but not at the HM loci (27Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (554) Google Scholar, 28Laroche T. Martin S.G. Gotta M. Gorham H.C. Pryde F.E. Louis E.J. Gasser S.M. Curr. Biol. 1998; 8: 653-656Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, silencing of different regions appears to involve non-identical regulatory mechanisms that have some common features. It is notable that many of the genes that affect silencing at telomeres and the HM loci are also involved in some aspects of DNA replication (29Ehrenhofer-Murray A.E. Kamakaka R.T. Rine J. Genetics. 1999; 153: 1171-1182PubMed Google Scholar, 30Dillin A. Rine J. Genetics. 1997; 147: 1053-1062Crossref PubMed Google Scholar, 31Ehrenhofer-Murray A.E. Gossen M. Pak D.T. Botchan M.R. Rine J. Science. 1995; 270: 1671-1674Crossref PubMed Scopus (52) Google Scholar). However, a number of studies suggest that DNA replication itself is not required for silencing (29Ehrenhofer-Murray A.E. Kamakaka R.T. Rine J. Genetics. 1999; 153: 1171-1182PubMed Google Scholar, 30Dillin A. Rine J. Genetics. 1997; 147: 1053-1062Crossref PubMed Google Scholar, 32Kirchmaier A.L. Rine J. Science. 2001; 291: 646-650Crossref PubMed Scopus (108) Google Scholar). For example, analysis of the two components of ORC, ORC2 and ORC5, showed that their roles in silencing and replication are separable (30Dillin A. Rine J. Genetics. 1997; 147: 1053-1062Crossref PubMed Google Scholar, 31Ehrenhofer-Murray A.E. Gossen M. Pak D.T. Botchan M.R. Rine J. Science. 1995; 270: 1671-1674Crossref PubMed Scopus (52) Google Scholar). Another transcriptional repression phenomenon was recently described for Ty1-transposable elements (33Jiang Y.W. Genes Dev. 2002; 16: 467-478Crossref PubMed Scopus (36) Google Scholar, 34Morillon A. Benard L. Springer M. Lesage P. Mol. Cell. Biol. 2002; 22: 2078-2088Crossref PubMed Scopus (49) Google Scholar). Ty elements are dormant under normal conditions but under stress can be induced to produce as much as 10% of the total RNA (33Jiang Y.W. Genes Dev. 2002; 16: 467-478Crossref PubMed Scopus (36) Google Scholar, 35Morillon A. Springer M. Lesage P. Mol. Cell. Biol. 2000; 20: 5766-5776Crossref PubMed Scopus (55) Google Scholar, 36Bradshaw V.A. McEntee K. Mol. Gen. Genet. 1989; 218: 465-474Crossref PubMed Scopus (103) Google Scholar, 37Staleva Staleva L. Venkov P. Mutat. Res. 2001; 474: 93-103Crossref PubMed Scopus (39) Google Scholar). Re-establishment of the silent state in all genome Ty1 elements is mediated by a co-suppression mechanism, which requires expression of Ty1 from a high copy plasmid and is counteracted by the SWI/SNF chromatin-modifying complex and the histone acetyltransferase SAGA (33Jiang Y.W. Genes Dev. 2002; 16: 467-478Crossref PubMed Scopus (36) Google Scholar, 34Morillon A. Benard L. Springer M. Lesage P. Mol. Cell. Biol. 2002; 22: 2078-2088Crossref PubMed Scopus (49) Google Scholar). As in TPE, Ty suppression is epigenetic such that in a single cell all or none of the Ty elements are expressed (33Jiang Y.W. Genes Dev. 2002; 16: 467-478Crossref PubMed Scopus (36) Google Scholar, 34Morillon A. Benard L. Springer M. Lesage P. Mol. Cell. Biol. 2002; 22: 2078-2088Crossref PubMed Scopus (49) Google Scholar). In this report we analyzed global gene expression in four mcm5 mutants. We found that a considerable number of genes in the vicinity of telomeres and Ty elements were up-regulated at the restrictive temperature in two of the mutants. This up-regulation correlated with chromatin remodeling of the affected genome regions and reversal of TPE. Yeast Strains and Plasmids—The strains used in this study are listed in Table I. Complemented meroploid mcm5-1::MCM5, mcm5-2::MCM5, mcm5-3::MCM5, and mcm5-461::MCM5 were produced by transforming the corresponding mutant mcm5 strains with YIp122CDC46 (38Gauthier L. Dziak R. Kramer D.J. Leishman D. Song X. Ho J. Radovic M. Bentley D. Yankulov K. Genetics. 2002; 162: 1117-1129PubMed Google Scholar) linearized by BspHI to produce tandem MCM5 duplications. Positive clones were selected on SC/-Leu plates and then on YPD (1% yeast extract, 2% peptone, 2% glucose) plates at 37 °C. Telomere reporter strains, mcm5-461-VIIL-URA3-tel, mcm5-461-VR-URA3-tel, mcm5-461::MCM5-VIIL-URA3-tel, and mcm5-461::MCM5-VR-URA3-tel were produced by transforming mcm5-461::MCM5 and mcm5-461 with YIpADH4UCAIV (39Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1134) Google Scholar) linearized by SalI and EcoRI or by YIpUCAV (39Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1134) Google Scholar) linearized by EcoRI, respectively, and selecting on SC/-Ura. URA3 is inserted next to the partially truncated telomeric TG1–3 repeats of the left arm of chromosome VII (VIIL-URA3-tel) and the right arm of chromosome V (VR-URA3-tel), respectively (39Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1134) Google Scholar). Positive clones were streaked on SC/-Ura, FOA, and SC/-Leu plates at 30 °C and on YPD medium at 37 °C to select for strains that exhibit true TPE and display the expected phenotypes. The pAS-TRA1 plasmid (2 μm, TRP1, TRA1) was described in a previous study (40Saleh A. Schieltz D. Ting N. McMahon S.B. Litchfield D.W. Yates 3rd, J.R. Lees-Miller S.P. Cole M.D. Brandl C.J. J. Biol. Chem. 1998; 273: 26559-26565Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).Table IYeast strainsStrainGenotype/phenotypemcm5-1 aThese strains are derivatives of 8534-8C (43, 44).mcm5-1 ura3-52 leu2-3, 112 MATα/Ts at 37 °Cmcm5-1::MCM5mcm5-1::MCM5::LEU2 ura3-52 leu2-3, 112 MATαmcm5-2 aThese strains are derivatives of 8534-8C (43, 44).mcm5-2 ura3-52 leu2-3, 112 his3-200 MATα/Ts at 37 °Cmcm5-2::MCM5mcm5-2::MCM5::LEU2 ura3-52 leu2-3, 112 his3-200 MATαmcm5-3 aThese strains are derivatives of 8534-8C (43, 44).mcm5-3 leu2-3, 112 MATα/Ts at 37 °Cmcm5-3::MCM5mcm5-3::MCM5::LEU2 leu2-3, 112 MATαmcm5-461 bThis strain is identical to cdc46-1 in Refs. 43 and 44.mcm5-461 ura3-52 leu2-3, 112 ade2 lys2-801 MATα/Ts at 37 °Cmcm5-461::MCM5mcm5-461::MCM5::LEU2 ura3-52 leu2-3, 112 ade2 lys2-801 MATαa These strains are derivatives of 8534-8C (43Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar, 44Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (230) Google Scholar).b This strain is identical to cdc46-1 in Refs. 43Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar and 44Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (230) Google Scholar. Open table in a new tab Microarray Analysis of Gene Expression—Mutant mcm5 and complemented mcm5::MCM5 strains were grown in YPD at 30 °C or 37 °C to A 600 = 0.2–0.5 and harvested on crushed ice. Total RNA was isolated by the lithium chloride method and cDNA was synthesized from 10 μg of total RNA by reverse transcribing with SuperScript II (Stratagene) in the presence of amino-allyl dUTP. N-Hydroxysuccinimide Cy5 and Cy3 dyes (Amersham Biosciences) were coupled to the amine-modified cDNA according to the instructions of the manufacturer. Microarrays containing all 6200 ORFs from the S. cerevisiae genome were from the Microarray Center at OCI, Toronto, Canada. Hybridization was for 18 h at 37 °C. The microarrays were scanned with Axon GenePix 4000a microarray scanner. Differentially expressed genes were identified as 2-fold up- or down-regulated and clustered for n number of experiments with GeneSpring version 5.0 software (Silicon Genetics). Statistical Evaluation of the Data—The evaluation was performed according to the textbook "Introductory Statistics" (41Hassett N. Weiss M. Introductory Statistics. 3rd. Ed. Addison-Wesley, 1991Google Scholar) and was based on the information provided in mips.gsf.de/proj/yeast and Refs. 22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar and 42Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3899) Google Scholar. Northern Blot Analysis—Northern blots were performed with 10 μg of total RNA isolated from cells grown at 30 °C or 37 °C to A 600 = 0.2–0.5. Probes were synthesized by PCR from S. cerevisiae genomic DNA with primer pairs, which amplified fragments as follows: a 500-bp fragment from COS1, a 400-bp fragment from YFL068, a 460-bp fragment from HXK1, a 430-bp fragment from ACT1, a 330-bp fragment from DAN3, a 570-bp fragment from NCE103, and a 400-bp fragment from the Ty1B protein. The HXK1, ACT1, DAN3, NCE103, and Ty1B fragments recognize unique sequences in the yeast genome as confirmed by BLAST search. The COS1 probe shares significant homology with the conserved sub-telomeric COS1–8 genes. The YFL068 probe shares homology with 10 other sub-telomeric ORF sequences. The amplified fragments were synthesized by a second round of PCR to minimize the amount of genomic DNA in the sample and then labeled by random primer. The Northern protocol, PCR conditions, and primer sequences are available upon request. Telomere Position Effect Assay—The TPE assay was performed as described earlier (39Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Abstract Full Text PDF PubMed Scopus (1134) Google Scholar) with some modifications. Briefly, three independent isolates of mcm5-461-VIIL-URA3-tel, mcm5-461-VR-URA3-tel, mcm5-461::MCM5-VIIL-URA3-tel, and mcm5-461::MCM5-VR-URA3-tel were streaked on YPD, Sc/-Ura, and FOA plates to confirm variegated expression of URA3 and then grown for about 12 generations in YPD liquid cultures at 30 °C or 37 °C. Serial 1:10 dilutions of these cultures were spotted on YPD, SC/-Ura, and FOA plates and grown at 30 °C until colonies were visible. The colonies were counted and average percent of FOAR cells was estimated. Micrococcal Nuclease Sensitivity Assay—Mutant mcm5 and complemented mutant strains were grown in 150 ml of YPD to A 600 = 0.5–1 and spheroplasts were prepared by Zymolyase treatment for 10–15 min at 37 °C. Spheroplasts were re-suspended by vortexing for 15 s in 2 ml of lysis/MN solution (30 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1 mg/ml bovine serum albumin, 10 mm MgCl2, 10 mm CaCl2, 100 units/ml micrococcal nuclease (Worthington), 1 μg/ml pepstatin, 1 μg/ml leupeptin, 2 μg/ml aprotinin) at 37 °C. 400-μl aliquots were removed at different time points and added to 100 μl of STOP solution (3 m NaCl, 3% SDS, 100 mm EDTA, 20 mm EGTA) pre-equilibrated at 60 °C. DNA was isolated, and 10 μg were analyzed by Southern blot with the same probes that were used for Northern analysis. Analysis of Differential Gene Expression in mcm5 Strains— An earlier analysis of gene expression of the mcm5-461 strain has indicated that several COS genes, which are positioned next to telomeres, were up-regulated at 37 °C compared with 30 °C (Ref. 38Gauthier L. Dziak R. Kramer D.J. Leishman D. Song X. Ho J. Radovic M. Bentley D. Yankulov K. Genetics. 2002; 162: 1117-1129PubMed Google Scholar and data not shown). It was unclear if the observed effect was determined by the position of the COS genes or it is a normal response to elevated temperature. To systematically correlate MCM5 function to gene expression we selected four previously characterized mcm5 strains (43Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar, 44Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (230) Google Scholar) and produced isogenic complemented strains (Table I) by inserting MCM5-LEU2 alleles in the corresponding mcm5 alleles. mcm5-2 and mcm5-461 displayed significant temperature sensitivity at 37 °C relative to their corresponding complemented strains (Fig. 1). mcm5-1 showed intermediate and mcm5-3 showed mild Ts phenotypes, respectively (Fig. 1). All complemented strains grew indistinguishably from unrelated wt strains at both 30 °C and 37 °C (data not shown). We used these strains to conduct micro-array analysis of global gene expression. Two independent experiments were performed with each mutant versus its complemented strain at both 30 °C and 37 °C. We defined differentially expressed genes as 2-fold up- or down-regulated. Using this criterion we looked for common ORFs in different experiments by the clustering algorithm of the GeneSpring version 5.0 software. These analyses identified no consistent differences between complemented and mutant strains at 30 °C (analysis not included). At the same time, the same criteria pointed to considerable similarities in the expression patterns of mcm5-1 and mcm5-461 versus their isogenic complemented counterparts at 37 °C (Table II). In these strains we found 85 genes that were up-regulated and five genes that were down-regulated in three out of the four experiments. Comparison of all mcm5 mutants at 37 °C did not reveal other clusters of common genes (analysis not included). There were no significant similarities between mcm5-2 and mcm5-3 (analysis not included). In summary, we observed a common pattern of differentially expressed genes in the mcm5-1 and the mcm5-461 strains at the restrictive temperature. It is important to note that mcm5-1 and mcm5-461 were identified in different screens using different parent strains (43Maine G.T. Sinha P. Tye B.K. Genetics. 1984; 106: 365-385Crossref PubMed Google Scholar, 44Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (230) Google Scholar).Table IIDifferentially expressed genes in mcm5–1 and mcm5–461Systematic nameCommon nameI. Genome positionII. Next to ARSIII. Cell cycle regulationIV. Normalized values in individual experimentsAt least 2× up-regulated ORFs in mcm5 versus complemented strainsYAR010CTY, CEN4.225.02.61.5YBL005W-APDR3TY4.820.02.92.2YBL099WATP1TY3.12.01.42.1YBL101CECM21tel, TY2.72.22.10.9YBL111CTELG12.97.12.01.7YBR012W-BTY4.516.74.52.7YBR068CBAP22.12.32.12.2YBR072WHSP2620.012.516.73.1YBR080CSEC18tRNA3.42.65.02.0YBR218CPYC22.63.42.6YBR289WSNF5tel3.02.52.21.7YBR301WDAN3TEL3.12.72.9YCL020WTY2.63.82.91.7YCL040WGLK1M/G14.55.08.30.9YDL124W3.64.04.81.1YDL248WCOS7TEL2.44.02.5YDR077WSED1G2/M5.310.03.33.4YDR171WHSP42TYPro-ARS3.81.68.30.8YDR298CATP52.41.12.71.5YDR342CHXT7LTRFalse ARSM/G11.95.62.6YDR366CTY3.87.73.62.4YEL039CCYC73.60.63.11.2YEL060CPRB1S6.36.74.31.1YEL077CTELPro-ARSG14.28.32.21.9YER004WCEN2.62.02.41.5YFL014WHSP122.03.71.72.4YFL062WCOS4TEL2.64.31.9YFL068WTEL2.32.42.31.9YFR053CHXK1telPro-ARS4.02.410.01.1YGL055WOLE1tRNA, LTRM/G12.26.30.72.3YGL248WPDE12.32.72.01.2YGR161CTY3.12.42.81.2YGR180CRNR4tel, tRNA11.15.94.21.7YGR294WTEL0.62.12.62.0YGR295CCOS6TELFalse ARS2.33.63.32.4YGR296WYRF1-3TELG12.69.12.51.8YHL021CtRNA, LTR,Pro-ARS3.33.24.50.7YHL050CTELG12.93.01.32.5YHR030CSLT2S2.22.32.42.0YHR087W6.72.29.11.1YHR218WTELG12.35.92.41.5YIL107CPFK262.02.73.11.2YJL026WRNR210.03.65.61.8YJL158CCIS3tRNAS/G22.63.11.42.5YJL159WHSP150tRNAM/G17.12.73.71.9YJR026WTY3.620.02.31.5YJR029WTY2.14.52.32.6YJR045CSSC1tRNA, LTR4.23.63.00.8YKL067WYNK1tRNA, LTRG12.31.12.02.0YKL109WHAP4tel5.02.62.3YKL151CtRNA, LTRM/G13.01.82.70.8YKL163WPIR3M/G14.85.94.82.3YLL026WHSP104tRNA, LTR,False ARS3.32.17.70.8YLR121CYPS3G12.73.81.91.6YLR142WPUT1tRNAS/G22.22.32.41.1YLR155CASP3-1rRNA, TY2.02.22.81.3YLR194CTYM/G11.93.72.91.9YLR216CCPR62.53.32.00.7YLR327CtRNA2.43.32.21.4YLR334Ctel, LTR2.92.22.52.5YLR391WSSR1tel2.83.33.62.7YLR466WYRF1-4TELFalse ARSM/G12.96.32.01.5YML100WTSL12.94.35.61.1YML128CMSC1TEL2.22.04.35.6YML132WCOS3TEL1.63.32.82.2YMR051CTY, tRNA3.811.13.41.9YMR105CPGM2tRNAFalse ARS5.02.64.80.8YMR169CALD31.72.42.80.8YMR186WHSC82Pro-ARS4.22.42.20.6YMR273CZDS1LTR2.91.62.22.3YNL036WNCE103TY2.64.35.32.2YNL055CPOR1TY4.84.00.92.6YNL160WYGP1M/G13.24.02.60.9YNL192WCHS1Pro-ARSM/G13.34.82.21.7YNL208WG12.42.94.52.0YNL336WCOS1TELPro-ARS3.23.45.32.3YOL016CCMK2LTRG12.62.02.21.1YOL053C-ADDR2LTR5.03.316.72.9YOR374WALD4TEL2.61.82.61.2YOR385WTEL3.32.82.21.0YPL058CPDR12TYG2/M2.93.13.11.4YPL154CPEP4False ARS3.33.43.31.1YPL271WATP15tel2.73.42.11.5YPR160WGPH1TYG11.71.42.65.9YPR204WTELM/G12.710.01.12.0At least 2× down-regulated ORFs in mcm5 versus complemented strainsYKL081WTEF40.30.30.80.1YOR333C0.30.40.20.7YDL051WLHP1tRNA0.50.30.90.3YHR016CYSC84tRNA, LTR0.10.10.11.2YCRX04W0.40.90.40.3 Open table in a new tab Characteristics of the Up-regulated Genes in the mcm5–1 and mcm5-461 Strains—We defined several genome elements and areas of interest and examined the positions of the up-regulated genes in the mcm5-1 and mcm5-461 strains relative to these elements (Table II, column I). We also examined if the ORFs are immediately next to a pro-ARS (binds MCM·ORC proteins and has ARS activity (22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar)) or a false ARS (binds MCM·ORC proteins but has no ARS activity (22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar)) sites (Table II, column II). Because some mcm mutants arrest in early S or before M phase of the cell cycle, we examined if the listed ORFs were previously reported as cell cycle-regulated genes (42Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3899) Google Scholar) (Table II, column III). We noticed a high representation of ORFs within 5 kb of Y′ or X telomere elements, within 3 kb of Ty and within 2 kb of solo Ty-LTR elements or tRNA genes as well as genes whose expression is at its peak in M/G1 and G1 phases of the cell cycle. It is worthwhile mentioning that a group of eight genes within 5 kb of telomeres, five genes within 3 kb of Ty elements, and nine other genes were up-regulated in two of the four experiments but did not show a value in the other two as determined by the GeneSpring software. These genes were excluded from future analyses. To evaluate the significance of this higher representation of genes we estimated the total number of ORFs in the analyzed genome areas (Table III, column I) and calculated their expected random frequency (E) (Table III, column III). We also calculated the expected random frequency (E) of genes next to pro-ARS and false ARS elements (22Wyrick J.J. Aparicio J.G. Chen T. Barnett J.D. Jennings E.G. Young R.A. Bell S.P. Aparicio O.M. Science. 2001; 294: 2357-2360Crossref PubMed Scopus (338) Google Scholar) and cell cycle-regulated genes (42Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3899) Google Scholar) (Table III, column III). The observed frequency (O)of all these ORFs was calculated in Table III (column IV). Finally, we calculated the difference between expected and observed frequency as (O – E)2/E (Table III, column V). A value close to 0 indicates random distribution. We observed significantly higher than expected frequency for ORFs within 5 kb of telomeres and within 3 kb of Ty elements, respectively (Table III, column V). The representatives of these two main groups of ORFs constitute 41% of the up-regulated genes in the two mcm5 strains and 4% of the total ORFs in the genome. ORFs within 1.5 kb of tRNA genes, solo Ty-LTR elements and 3 kb of rRNA genes showed moderate increase of the observed relative to the expected frequency, whereas ORFs within 5–20 kb from telomeres, 5 kb from centromeres or immediately next to pro-ARS or false ARS elements appeared at the random frequency (Table III, column V). In the group of cell
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