* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Chromosome segregation: Samurai separation
Survey
Document related concepts
Histone acetyltransferase wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Protein moonlighting wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
X-inactivation wikipedia , lookup
Point mutation wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Transcript
Dispatch R531 Chromosome segregation: Samurai separation of Siamese sisters Michael Glotzer How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids. Address: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. E-mail: [email protected] Current Biology 1999, 9:R531–R534 http://biomednet.com/elecref/09609822009R0531 © Elsevier Science Ltd ISSN 0960-9822 Chromosomes are segregated with high fidelity; in yeast, chromosome loss occurs only about once per 105–106 cell divisions [1,2]. The machinery responsible for this critical task is of course the mitotic spindle, a microtubule-based, bipolar structure, in which microtubules attach to chromosomes at a specialized site called the kinetochore. It has long been appreciated that, during mitosis, sister chromatids — the DNA molecules that arise by replication of a single chromosome — align on the mitotic spindle by the binding of sister kinetochores to microtubules growing from opposite poles of the mitotic spindle. A combination of forces contributed by microtubule dynamics and microtubule-based molecular motors balances the chromosomes midway between the poles of the mitotic spindles. This structural organization is well suited to the equal partitioning of the genome. Moreover, defects in various components are thought to contribute to the development of certain cancers [3]. I shall focus here on the connection between sister chromatids and how it is dissolved in an orderly fashion at anaphase. In eukaryotes there is a temporal delay between the time of replication of the genome (S phase) and the time of its segregation (M phase). To ensure that chromosomes are segregated equally, sister chromatids are linked to each other from the time of their synthesis to the time of their segregation. Although mechanisms do exist that allow similar chromosomes to find each other, as evidenced by the pairing of homologous chromosomes during meiosis, during mitotic cell cycles linkage is established at the time of DNA replication and is maintained between sister chromatids until anaphase [4]. In recent years, the nature of this linkage has been under intense scrutiny, and a recent study has uncovered a surprising twist to the story of how the linkage is dissolved at anaphase. Anaphase onset is not only the abrupt moment at which sister chromatids separate from each other, but it is also the moment at which the cell begins to transit from a mitotic state to an interphase state. This transition is, in part, mediated by the destruction of cyclin, the regulatory subunit of the central regulator of mitosis, a kinase complex containing cyclin B and cyclin-dependent kinase 1 (Cdk1) [5]. Early experiments indicated that sister separation and cyclin destruction are independently regulated by multiubiquitination of specific substrates by the anaphase promoting complex (APC) and their subsequent destruction by the 26S proteasome. Specific inhibition of cyclin proteolysis or general inhibition of the APC both confer a mitotic arrest phenotype. But while inhibition of the APC causes a metaphase arrest, inhibition of cyclin proteolysis allows chromosomes to separate from each other [6,7]. This suggests that a protein other than cyclin must be degraded in an APCdependent fashion to allow anaphase to occur. One interpretation of these results is that this protein forms a proteinaceous ‘glue’ that holds sister chromatids together. An equally plausible explanation is that this protein is a soluble inhibitor of anaphase, and that its APC-mediated destruction indirectly permits dissolution of the bond that holds sisters chromatids together. Over the last several years the simpler interpretation, the ‘glue’ hypothesis, has suffered a significant setback, and the alternative ‘anaphase inhibitor’ hypothesis has gained considerable support. A mutation was identified in a budding yeast gene called PDS1 that caused premature dissociation of sister chromatids (hence the gene name) [8,9]. The encoded Pds1 protein contains a destruction box characteristic of APC substrates; the protein was found to be degraded at the metaphase-to-anaphase transition, and expression of a non-degradable version blocked cells in metaphase [10]. Moreover, Pds1 is the only protein that must be targeted for degradation by the APC to allow anaphase to occur [11]. However, PDS1 is not an essential gene in budding yeast, nor is its product associated with chromosomes, as one would expect if it were a protein that physically holds sisters together. What does Pds1 do to prevent sisters from separating? And what holds them together, if it is not Pds1? A clue to the first question came in the form of a protein, Esp1, that was found to be associated with Pds1 in budding yeast cells [11]. Esp1 is a 180 kDa protein with a conserved carboxyterminal domain [12]. ESP1 function is required for the separation of sister chromatids and its activity is negatively regulated by Pds1 [11]. Thus, for anaphase to occur, Pds1 is degraded by APC-mediated ubiquitination, allowing R532 Current Biology, Vol 9 No 14 Esp1 to become active. Esp1, like most of the known regulators of the cell cycle, is quite highly conserved; Esp1 homologues can be readily recognized across the animal kingdom. The sequence conservation reflects an underlying functional conservation, as the fission yeast homolog of Esp1, Cut1, is also required for sister separation and it associates with a protein, Cut2, that is functionally analogous to Pds1 [13]. Remarkably, despite their functional conservation Pds1 and Cut2 are only 13% identical in sequence, which is not usually considered statistically significant. This lack of sequence conservation greatly hampered the identification of vertebrate equivalents of Pds1. A lead has come, however, from the recent identification of a Xenopus protein, xPDS1, on the basis of its instability in mitotic, but not interphase, extracts [14]. The xPDS1 protein is about 30% identical in sequence to a human protein encoded by a gene called pituitary tumor transforming gene (PTTG). Each of these vertebrate proteins is only about 15% identical in sequence to yeast Pds1, but their genes appear to be functional homologs of PDS1 and Cut2. PTTG associates with hESP1 and nondegradable mutant forms of xPDS1 cause dominant mitotic arrest. Thus, in both vertebrates and yeast, anaphase onset requires the destruction of a rapidly evolving protein that acts as an inhibitor of Esp1. What does active Esp1 actually do? The answer to this question requires a digression into the nature of the linkage that holds sister chromatids together. In budding yeast, sister chromatid linkage, or cohesion, is mediated by a complex of proteins that contains, at the least, two Smc proteins and two additional conserved components, Scc3 and Scc1/Mcd1, which together form the so-called cohesin complex [13–15]. Smc proteins, named for their involvement in the structural maintenance of chromosomes, are required for chromosome condensation, recombination and dosage compensation [16]. These proteins are a structural curiosity. Their sequences reveal three distinct regions: an amino-terminal region that contains part of an NTP-binding motif; a central coiledcoil region; and a carboxy-terminal region that contains the remaining parts of the NTP-binding motif and a domain that binds DNA [17]. Electron microscopy showed that Smc proteins form antiparallel dimers with a central hinge and two identical terminal globular domains that bind nucleotide and DNA [18]. Though still not fully established, the cohesin complex fits the bill for a proteinaceous link between sister chromatids. It is required from S phase until M phase, and during this time it is bound to chromosomes. The complex then dissociates at the onset of anaphase in a reaction that depends on Esp1. In order to examine how Esp1 causes cohesin dissociation, an in vitro assay was developed in which cohesins bound to chromatin could be mixed with soluble extracts from cells overproducing Esp1. Using this assay, Esp1-containing extracts were found to cause Scc1 to dissociate from chromatin [19]. The surprising result was that one cohesin subunit, Scc1, underwent a dramatic mobility shift of approximately 40 kDa in response to Esp1 activity. Such a significant mobility shift suggested a proteolytic event. Two controls suggested that this processing was specific: firstly, the Scc1 associated with the chromatin was much more sensitive to the processing event than soluble Scc1; and secondly, the reaction was inhibited by Pds1. The nature of the processing event was established by analysis of a derivative of Scc1 carrying distinct epitope tags at either end. When this substrate was subjected to Esp1containing extracts, the two epitopes migrated at distinct mobilities, confirming that Esp1 indeed induced proteolytic cleavage of Scc1. A cleaved product of the appropriate molecular weight could also be observed in vivo when a synchronous culture of cells exited mitosis. These data suggest that the cleavage of Scc1 is stimulated by Esp1, raising the possibility that this cleavage might regulate separation of sister chromatids. To test this hypothesis, a version of Scc1 that could not be cleaved was generated and tested in vivo [19]. First, the site of cleavage was identified by sequencing the carboxy-terminal fragment of Scc1. An arginine residue immediately upstream of the first residue of the carboxy-terminal fragment was mutated, and the resulting mutant protein inducibly expressed in cells. Yeast cells expressing this mutant protein could still grow quite well; biochemical analysis, however, revealed that this protein was still subject to Esp1-induced cleavage, albeit at a site aminoterminal to the previously identified cleavage site. Sequence analysis revealed a second sequence motif very similar to the initial cleavage site. A double mutant form of Scc1 was therefore designed to abolish both cleavage sites. The double-mutant protein was found to be resistant to Esp1-mediated cleavage and toxic to yeast cells in which it was expressed. In cells expressing the double-mutant protein, sister chromatids failed to separate and the cohesin complex remained associated with chromosomes. The double-mutant protein could, however, mediate cohesion between sister chromosomes, so it appears to be specifically defective in the dissociation step at anaphase. Esp1-induced cleavage of Scc1 is thus an essential step in the separation of sister chromatids at the metaphase-to-anaphase transition. The glue hypothesis has thus undergone a dramatic renaissance, only the mechanism and its regulation is far more complex than previously thought (Figure 1). At the metaphase-to-anaphase transition, ubiquitin-mediated proteolysis of a non-cyclin protein, Pds1, is required for sister chromatid separation. Pds1 acts to inhibit Esp1, which in Dispatch R533 Figure 1 A model of the reactions required for the separation of sister chromatids at the metaphase-to-anaphase transition. The anaphase promoting complex (APC) ubiquitinates Pds1, thus targeting it for degradation and relieving Esp1 from inhibition. Subsequently, Esp1 induces the proteolytic cleavage of Scc1 and allows the mitotic spindle to separate the sister chromatids. Pds1 Esp1 Smc1+ Smc3 Scc1 Scc3 APC-mediated Pds1 degradation Esp1 Current Biology turn either directly or indirectly causes one member of the cohesin complex, Scc1, to be chopped into two parts, allowing the sisters to separate. The remaining components of the cohesin complex may subsequently dissociate after anaphase. These results raise several important questions. Firstly, how does Esp1 induce Scc1 proteolysis — is Esp1 itself the Scc1 protease? Analysis of the Esp1 sequence has failed to reveal any hallmarks of proteases; it is, however, possible that Esp1 is a novel protease, or that the degree of conservation is insufficient to allow recognition of a known class of proteases. The answer to this question will likely be uncovered by biochemical purification of the Scc1 cleavage activity. Secondly, have these studies in budding yeast revealed a conserved mechanism for regulation of anaphase? This is a very interesting question and several pieces of information would suggest it is a universal mechanism, though other data suggest otherwise. The Scc1 cleavage site is conserved in a Scc1-like protein, Rec8, required for sisterchromatid cohesion during meiosis in budding yeast, and in two related proteins of the fission yeast Schizosaccharomyces pombe, Rad21 and Rec8. The cleavage site is not, however, conserved in vertebrate Scc1. It is possible that the general mechanism — Esp1mediated cleavage of Scc1 — is conserved in vertebrate cells, but that the recognition site for cleavage has diverged beyond recognition. However, vertebrate Scc1 has been characterized in some detail, and it has been found to dissociate from chromosomes upon entry into mitosis, rather than at anaphase onset as in budding yeast [20]. In view of this difference, why should Esp1 be conserved, and why does a non-degradable Pds1 block anaphase onset in vertebrate cells? The explanation may be that a small fraction of Scc1 remains on chromosomes, or that there is a second Scc1-like protein that has not yet been identified that remains on chromosomes until Esp1 induces its cleavage. These questions are sure to be answered shortly, but the answers to a host of other interesting questions may require a longer wait. These include questions such as why are the anaphase inhibitors evolving so rapidly? Is Pds1 the only regulator of Esp1 activity? Are cohesins the sole cellular substrates of Esp1-mediated cleavage? While these regulatory issues are of great interest, the structural aspects of chromosome cohesion and separation are perhaps even more provocative and challenging. As the recent discovery of a large duplex loop at eukaryotic telomeres [21] has so vividly demonstrated, there is often no substitute for direct visualization. How are chromosome cohesion sites arranged? Are they at the base of chromatin loops? How many cohesin complexes are present at each site? Does catenation of sister DNA molecules play any role in chromosome cohesion? Do all cohesin complexes need to be destroyed by cleavage of the Scc1 to allow sisters to separate? It will be satisfying to scrutinize the system that enables Siamese sisters to separate successfully. R534 Current Biology, Vol 9 No 14 Acknowledgements I thank Jan-Michael Peters and Jola Glotzer for helpful comments on this dispatch. If you found this dispatch interesting, you might also want to read the April 1999 issue of References 1. Meeks-Wagner D, Hartwell LH: Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell 1986, 44:43-52. 2. Murray AW, Schultes NP, Szostak JW: Chromosome length controls mitotic chromosome segregation in yeast. Cell 1986, 45:529-536. 3. Lengauer C, Kinzler KW, Vogelstein B: Genetic instability in colorectal cancers. Nature 1997, 386:623-627. 4. Uhlmann F, Nasmyth K: Cohesion between sister chromatids must be established during DNA replication. Curr Biol 1998, 8:1095-1101. 5. Murray AW, Solomon MJ, Kirschner MW: The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 1989, 339:280-286. 6. Holloway SL, Glotzer M, King RW, Murray AW: Anaphase is initiated by proteolysis rather than by the inactivation of maturationpromoting factor. Cell 1993, 73:1393-1402. 7. Surana U, Amon A, Dowzer C, McGrew J, Byers B, Nasmyth K: Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J 1993, 12:1969-1978. 8. Yamamoto A, Guacci V, Koshland D: Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae. J Cell Biol 1996, 133:85-97. 9. Yamamoto A, Guacci V, Koshland D: Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Biol 1996, 133:99-110. 10. Cohen-Fix O, Peters JM, Kirschner MW, Koshland D: Anaphase initiation in Saccharomyces cerevisiae is controlled by the APCdependent degradation of the anaphase inhibitor Pds1p. Genes Dev 1996, 10:3081-3093. 11. Ciosk R, Zachariae W, Michaelis C, Shevchenko A, Mann M, Nasmyth K: An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 1998, 93:1067-1076. 12. McGrew JT, Goetsch L, Byers B, Baum P: Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae. Mol Biol Cell 1992, 3:1443-1454. 13. Funabiki H, Kumada K, Yanagida M: Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J 1996, 15:6617-6628. 14. Zou H, McGarry TJ, Bernal T, Kirschner MW: Identification of a vertebrate sister chromatid separation inhibitor encoded by a tumor transforming gene. Science 1999, in press. 15. Toth A, Ciosk R, Uhlmann F, Galova M, Schleiffer A, Nasmyth K: Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev 1999, 13:320-333. 16. Jessberger R, Frei C, Gasser SM: Chromosome dynamics: the SMC protein family. Curr Opin Genet Dev 1998, 8:254-259. 17. Akhmedov AT, Frei C, Tsai-Pflugfelder M, Kemper B, Gasser SM, Jessberger R: Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure. J Biol Chem 1998, 273:24088-24094. 18. Melby TE, Ciampaglio CN, Briscoe G, Erickson HP: The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge. J Cell Biol 1998, 142:1595-1604. 19. Uhlmann F, Lottspeich F, Nasmyth K: Sister chromatid separation at anaphase onset promoted by cleavage of the cohesin subunit Scc1p. Nature 1999, in press. 20. Losada A, Hirano M, Hirano T: Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev 1998, 12:1986-1997. 21. Griffith J, Comeau L, Rosenfield S, Stansel R, Bianchi A, Moss H, de Lange T: Mammalian telomeres end in a large duplex loop. Cell 1999, 97:503-514. Current Opinion in Genetics & Development which included the following reviews, edited by James T Kadonaga and Michael Grunstein, on Chromosomes and expression mechanisms: RNA polymerase II as a control panel for multiple coactivator complexes Michael Hampsey and Danny Reinberg Coactivator and corepressor complexes in nuclear receptor function Lan Xu, Christopher K Glass and Michael G Rosenfeld Chromatin-modifying and -remodeling complexes Roger D Kornberg and Yahli Lorch Activation by locus control regions? Frank Grosveld DNA methylation and chromatin modification Huck-Hui Ng and Adrian Bird Mechanisms of genomic imprinting Camilynn I Brannan and Marisa S Bartolomei Histone acetylation and cancer Sonia Y Archer and Richard A Hodin Modifying chromatin and concepts of cancer Sandra Jacobson and Lorraine Pillus Chromatin assembly: biochemical identities and genetic redundancy Christopher R Adams and Rohinton T Kamakaka Stopped at the border: boundaries and insulators Adam C Bell and Gary Felsenfeld Nuclear compartments and gene regulation Moira Cockell and Susan M Gasser Centromere proteins and chromosome inheritance: a complex affair Kenneth W Dobie, Kumar L Hari, Keith A Maggert and Gary H Karpen Telomeres and telomerase: broad effects on cell growth Carolyn M Price In vivo methods to analyze chromatin structure Robert T Simpson The full text of Current Opinion in Genetics & Development is in the BioMedNet library at http://BioMedNet.com/cbiology/gen