- Email: [email protected]
CENP-A Targeting Moves a Step Back
Molecular Cell
Previews CENP-A Targeting Moves a Step Back Richard E. Baker1,* 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.006
In a recent issue of Molecular Cell, Pidoux et al. (2009) and Williams et al. (2009) identify S. pombe Scm3 as the proximate factor in the Cnp1/CENP-A deposition pathway, providing a direct connection to centromerelocalized Mis16-Mis18.
Centromeres are the chromosomal sites at which microtubules attach to orchestrate accurate segregation of the cell’s DNA during mitosis and meiosis. Centromere DNAs vary widely in size and sequence, and except for the simple centromeres of some budding yeasts, centromere identity is determined with no absolute dependence on DNA sequence. Instead, centromere identity is established by a unique chromatin structure: active centromere DNA is packaged by nucleosomes containing a variant of histone H3 known as centromeric H3 (CenH3; CENP-A in mammals, Cnp1 in S. pombe, Cse4 in S. cerevisiae). A fundamental, unanswered question in centromere biology is how CenH3 is maintained specifically in centromere chromatin and excluded at other genomic loci. In a recent issue of Molecular Cell, Pidoux et al. (2009) and Williams et al. (2009) report characterization of S. pombe Scm3 (Scm3Sp). Their findings identify Scm3Sp as the proximate factor in the S. pombe CenH3 deposition pathway and raise new questions about the structure of CenH3 nucleosomes. Originally discovered in S. cerevisiae, Scm3 orthologs are widely conserved in fungi (Camahort et al., 2007; Mizuguchi et al., 2007; Stoler et al., 2007). S. cerevisiae have ‘‘point’’ centromeres, about 125 bp in length and consisting of three conserved DNA sequence elements (CDEs). Centromere identity is determined primarily by CDEIII, a 25 bp palindromic sequence recognized by the sequencespecific DNA-binding factor CBF3, whose binding is a prerequisite for assembly of a single Cse4-containing nucleosome that forms over CDEII (Furuyama and Biggins, 2007). S. cerevisiae Scm3 (Scm3Sc) binds Cse4 directly and interacts
with Ndc10, a subunit of CBF3, linking Cse4 deposition to sequence-specific recognition of centromere DNA by CBF3 (Figure 1). But Scm3Sc appears to be more than just an ‘‘adapter.’’ In vitro, Scm3Sc displaces H2A-H2B dimers from Cse4 histone octamers, and in vivo, the core centromere chromatin appears to lack both H2A and H2B, leading to the radical proposal that Scm3Sc is a core constituent of unconventional Cse4 nucleosomes, replacing H2A-H2B (Mizuguchi et al., 2007). Although the properties of Scm3Sc explain CenH3 targeting in S. cerevisiae, point centromeres are atypical. To what extent can the function(s) of Scm3Sc be generalized to the vast majority of eukaryotes that have complex ‘‘regional’’ centromeres possessed of no conserved identifier sequence? Now come Pidoux et al. (2009) and Williams et al. (2009) to answer this question. S. pombe regional centromeres consist of a central core (cnt) of nonrepetitive sequence flanked by inverted repeats. The innermost repeats (imr) together with the cnt comprise the central domain into which Cnp1 (S. pombe CenH3) is incorporated (Figure 1). Pidoux et al. (2009) identified Scm3Sp as the gene product of sim1, one of several sim mutants isolated in a previous genetic screen (Pidoux et al., 2003). Williams et al. (2009) used reverse genetics. Noticing that the uncharacterized open reading frame SPAPB1A10.02 encodes a protein with sequence similarity to Scm3Sc, they produced and analyzed tagged and temperature-sensitive alleles of the gene to deduce its function. Key results from both groups are in remarkable agreement and build a convincing case that Scm3Sp is a recruitment factor for Cnp1 at centromeres.
Coimmunoprecipitation, yeast two-hybrid (Y2H), and affinity capture demonstrated in vivo interaction between Scm3Sp and Cnp1. GST-pulldowns in vitro showed the interaction to be direct (Pidoux et al., 2009). Microscopic localization of GFPtagged Scm3Sp revealed it localizes at centromeres, and chromatin immunoprecipitation (ChIP) showed Scm3Sp to be present in central domain chromatin. Critically, Cnp1 fails to localize at centromeres in scm3 mutants, which also exhibit severe chromosome segregation defects. Equally persuasive are results arguing that Scm3Sp is not a constituent of Cnp1 nucleosomes. Both groups made the striking finding that, whereas Cnp1 is present at the centromere continuously throughout the cell cycle, Scm3Sp is lost from centromeres at mitosis onset—when one might assume the integrity of Cnp1 nucleosomes to be most critical—and returns in late anaphase. Also, Scm3Sp centromere localization is retained in cnp1-1 mutants; thus, Scm3Sp localization is independent of Cnp1. That Cnp1 is released from chromatin by micrococcal nuclease treatment under conditions where Scm3Sp remains insoluble adds biochemical corroboration (Pidoux et al., 2009). This is not to say that Cnp1 nucleosomes are ‘‘normal.’’ ChIP experiments by both groups showed a relative lack of H2A and H2B associated with centromere central domain DNA. The H2A and H2B ChIP signals were similar to that of H3, correlating inversely with that of Cnp1. In scm3 mutants, where centromere enrichment of Cnp1 is lost, H2B and H3 occupancies increase, with H4 occupancy largely unchanged (Williams et al., 2009). The direct conclusion is that a majority of Cnp1 ‘‘nucleosomes’’
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 411
Molecular Cell
Previews
Figure 1. CenH3 Deposition in S. cerevisiae and S. pombe (A) Scm3Sc-bound Cse4 is localized to S. cerevisiae point centromeres through interaction with the Ndc10 subunit of the CDEIII-binding factor CBF3. There, it is assembled into an unconventional hexameric nucleosome (Mizuguchi et al., 2007). Scm3Sc displaces H2A-H2B from Cse4 histone octamers in vitro, but it is not known if this is the operative pathway in vivo. (B) The central domain of S. pombe regional centromeres consists of the central core (cnt) and innermost repeats (imr). The outer repeats (otr) are packaged into heterochromatin. Cnp1 nucleosomes, possibly lacking H2A and H2B, are present in central domain chromatin. Scm3Sp mediates the interaction between Cnp1 and the centromere-bound Mis16-Mis18 complex, which, along with Scm3Sp, dissociates from centromeres at metaphase and returns during late anaphase (Pidoux et al., 2009; Williams et al., 2009).
lack H2A-H2B dimers. Another possibility is that Cnp1 nucleosomes are dynamic. The underrepresentation of H2A-H2B dimers would reflect their ‘‘last on-first off’’ status
in the assembly/disassembly process, causing inefficient crosslinking. More work will be required for a definitive explanation, but the data are quite intriguing in light of
412 Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc.
recent reports of unconventional CenH3 nucleosomes in S. cerevisiae and D. melanogaster (Dalal et al., 2007; Mizuguchi et al., 2007). If Scm3Sp is a recruitment factor and not part of Cnp1 nucleosomes, how is Scm3Sp itself localized to centromeres? The Mis16-Mis18 complex is the most upstream factor required for Cnp1 deposition (Hayashi et al., 2004). The cell-cycledependent localization of Scm3Sp mirrors that of Mis18 (Fujita et al., 2007), and both Pidoux et al. (2009) and Williams et al. (2009) find that Scm3Sp is mislocalized in mis16 and mis18 mutants. Using GST pulldown, Pidoux et al. (2009) demonstrated a direct protein-protein interaction between Scm3Sp and Mis18 (but not Mis16), whereas Williams et al. (2009) found a Y2H interaction between Scm3Sp and Mis16 (but not Mis18). Additional analysis is required to resolve the discrepancy, but the combined evidence strongly suggests that Scm3Sp links Mis16-Mis18 and Cnp1 through direct protein-protein interaction (Figure 1). Scm3Sp localization is also dependent upon Mis6 and Sim4 (Pidoux et al., 2009; Williams et al., 2009); however, unlike Scm3Sp, the Mis6-Sim4 complex is present at centromeres constitutively, and its role in maintaining Scm3Sp is not known. Also unknown is whether delocalization of Mis16-Mis18-Scm3Sp during M phase is a prerequisite for Cnp1 assembly in the subsequent cell cycle, although cycle-dependent localization of Mis18 is conserved. Human Mis18, which is required for de novo CENP-A deposition, associates only briefly with centromeres between late anaphase and G1 (Fujita et al., 2007). Scm3 homologs are not found in vertebrates, but it is probable that functional analogs exist. Likely candidates would be proteins associated with Mis18, perhaps Mis18BP (Fujita et al., 2007). The identification by Pidoux et al. (2009) and Williams et al. (2009) of Scm3Sp as the proximate factor in Cnp1 targeting, linking Cnp1 to centromere-localized Mis16Mis18, is a major advance. Left unanswered is how Mis16-Mis18 finds it way to centromere DNA. Effort must now be directed backward in the pathway, toward elucidating the mechanism of Mis16Mis18 localization and its cell-cycle regulation.
Molecular Cell
Previews REFERENCES Camahort, R., Li, B., Florens, L., Swanson, S.K., Washburn, M.P., and Gerton, J.L. (2007). Mol. Cell 26, 853–865. Dalal, Y., Wang, H., Lindsay, S., and Henikoff, S. (2007). PLoS Biol. 5, e218. 10.1371/journal.pbio. 0050218. Fujita, Y., Hayashi, T., Kiyomitsu, T., Toyoda, Y., Kokubu, A., Obuse, C., and Yanagida, M. (2007). Dev. Cell 12, 17–30.
Furuyama, S., and Biggins, S. (2007). Proc. Natl. Acad. Sci. USA 104, 14706–14711. Hayashi, T., Fujita, Y., Iwasaki, O., Adachi, Y., Takahashi, K., and Yanagida, M. (2004). Cell 118, 715–729. Mizuguchi, G., Xiao, H., Wisniewski, J., Smith, M.M., and Wu, C. (2007). Cell 129, 1153–1164. Pidoux, A.L., Richardson, W., and Allshire, R.C. (2003). J. Cell Biol. 161, 295–307.
Pidoux, A.L., Choi, E.S., Abbott, J.K.R., Liu, X., Kagansky, A., Castillo, A.G., Hamilton, G.L., Richardson, W., Rappsilber, J., He, X., and Allshire, R.C. (2009). Mol. Cell 33, 299–311.
Stoler, S., Rogers, K., Weitze, S., Morey, L., Fitzgerald-Hayes, M., and Baker, R.E. (2007). Proc. Natl. Acad. Sci. USA 104, 10571–10576.
Williams, J.S., Hayashi, T., Yanagida, M., and Russell, P. (2009). Mol. Cell 33, 287–298.
Closing the Feedback Loop: How Cells ‘‘Count’’ Telomere-Bound Proteins Neal F. Lue1,* 1Department of Microbiology and Immunology, W.R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.001
Telomere length homeostasis is thought to occur via a ‘‘protein-counting’’ mechanism whereby high numbers of telomere-bound proteins inhibit telomerase activity. In a recent issue of Molecular Cell, Hirano et al. (2009) delineate the molecular interactions that underlie the budding yeast protein-counting machinery. Telomeres are specialized nucleoprotein structures that maintain the integrity of eukaryotic chromosomal termini by protecting them from fusion and recombination and by promoting their replication (de Lange, 2005). In most organisms, telomeric DNA comprises short repetitive sequences that are rich in G residues on the 30 end-containing strand. These repeats are maintained by a ribonucleoprotein known as telomerase, which acts as an unusual reverse transcriptase (Autexier and Lue, 2006). Both telomerebinding proteins and telomerase are critical for the maintenance of telomere integrity through multiple cell divisions, which in turn is pivotal in supporting genome stability and promoting cellular life span. Not surprisingly, telomere lengths are regulated to within a defined size range in organisms ranging from yeast to man. The homeostatic system has been extensively analyzed and is best understood in the budding yeast Saccharomyces cerevisiae. Classic studies by Marcand et al. (1997) led to the proposal that telomere-
length regulation is achieved through a ‘‘protein-counting’’ mechanism whereby higher numbers of proteins bound by a longer telomere repeat tract ultimately inhibit telomerase activity at that particular telomere (Figure 1). The key elements of such a cis-acting inhibitory feedback loop are apparent from the outset: the factor being counted (the sensor), the factor or structure that transmits the count (the transducer), and the factor that elongates short telomeres (the effector, i.e., telomerase). Initially, the counted protein was thought to be Rap1, the major double strand telomere repeat binding factor. Subsequent studies by the Blackburn group suggested that Rif1 and Rif2, which are recruited to telomeres by a Rap1 C-terminal domain, can be ‘‘counted’’ by the cell in the absence of Rap1 (Levy and Blackburn, 2004). More recently, a series of elegant experiments by several labs pointed to the MRX (Mre11-Rad50-Xrs2) complex and Tel1 as plausible transducers of the system; these proteins are regulated by the Rif proteins, preferen-
tially localize to short telomeres, and positively promote telomerase action at telomeres (Bianchi and Shore, 2007; Hector et al., 2007; Sabourin et al., 2007; Tseng et al., 2006). Importantly, these studies identified a plausible set of interactions that relay the signal from the transducers to the effector. In essence, the MRX complex, once bound to short telomeres, enhances the localization of Tel1, which in turn phosphorylates Cdc13 and possibly other factors to promote telomerase recruitment and activity (Figure 1). What has been conspicuously missing, however, is a plausible molecular mechanism to relay the signals from any sensor (Rap1, Rif1, and Rif2) to the transducer (MRX and Tel1). This is where the recent study by Hirano et al. (2009) provides some satisfying answers. Using a system that induces a specific chromosome break adjacent to either telomere repeats or the bacterial Tet operator (TetO) repeats, Hirano et al. (2009) examined how tethering varying numbers of Rap1, Rif1, or Rif2 next to the DNA ends affects MRX and Tel1 localization.
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 413
Previews CENP-A Targeting Moves a Step Back Richard E. Baker1,* 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.006
In a recent issue of Molecular Cell, Pidoux et al. (2009) and Williams et al. (2009) identify S. pombe Scm3 as the proximate factor in the Cnp1/CENP-A deposition pathway, providing a direct connection to centromerelocalized Mis16-Mis18.
Centromeres are the chromosomal sites at which microtubules attach to orchestrate accurate segregation of the cell’s DNA during mitosis and meiosis. Centromere DNAs vary widely in size and sequence, and except for the simple centromeres of some budding yeasts, centromere identity is determined with no absolute dependence on DNA sequence. Instead, centromere identity is established by a unique chromatin structure: active centromere DNA is packaged by nucleosomes containing a variant of histone H3 known as centromeric H3 (CenH3; CENP-A in mammals, Cnp1 in S. pombe, Cse4 in S. cerevisiae). A fundamental, unanswered question in centromere biology is how CenH3 is maintained specifically in centromere chromatin and excluded at other genomic loci. In a recent issue of Molecular Cell, Pidoux et al. (2009) and Williams et al. (2009) report characterization of S. pombe Scm3 (Scm3Sp). Their findings identify Scm3Sp as the proximate factor in the S. pombe CenH3 deposition pathway and raise new questions about the structure of CenH3 nucleosomes. Originally discovered in S. cerevisiae, Scm3 orthologs are widely conserved in fungi (Camahort et al., 2007; Mizuguchi et al., 2007; Stoler et al., 2007). S. cerevisiae have ‘‘point’’ centromeres, about 125 bp in length and consisting of three conserved DNA sequence elements (CDEs). Centromere identity is determined primarily by CDEIII, a 25 bp palindromic sequence recognized by the sequencespecific DNA-binding factor CBF3, whose binding is a prerequisite for assembly of a single Cse4-containing nucleosome that forms over CDEII (Furuyama and Biggins, 2007). S. cerevisiae Scm3 (Scm3Sc) binds Cse4 directly and interacts
with Ndc10, a subunit of CBF3, linking Cse4 deposition to sequence-specific recognition of centromere DNA by CBF3 (Figure 1). But Scm3Sc appears to be more than just an ‘‘adapter.’’ In vitro, Scm3Sc displaces H2A-H2B dimers from Cse4 histone octamers, and in vivo, the core centromere chromatin appears to lack both H2A and H2B, leading to the radical proposal that Scm3Sc is a core constituent of unconventional Cse4 nucleosomes, replacing H2A-H2B (Mizuguchi et al., 2007). Although the properties of Scm3Sc explain CenH3 targeting in S. cerevisiae, point centromeres are atypical. To what extent can the function(s) of Scm3Sc be generalized to the vast majority of eukaryotes that have complex ‘‘regional’’ centromeres possessed of no conserved identifier sequence? Now come Pidoux et al. (2009) and Williams et al. (2009) to answer this question. S. pombe regional centromeres consist of a central core (cnt) of nonrepetitive sequence flanked by inverted repeats. The innermost repeats (imr) together with the cnt comprise the central domain into which Cnp1 (S. pombe CenH3) is incorporated (Figure 1). Pidoux et al. (2009) identified Scm3Sp as the gene product of sim1, one of several sim mutants isolated in a previous genetic screen (Pidoux et al., 2003). Williams et al. (2009) used reverse genetics. Noticing that the uncharacterized open reading frame SPAPB1A10.02 encodes a protein with sequence similarity to Scm3Sc, they produced and analyzed tagged and temperature-sensitive alleles of the gene to deduce its function. Key results from both groups are in remarkable agreement and build a convincing case that Scm3Sp is a recruitment factor for Cnp1 at centromeres.
Coimmunoprecipitation, yeast two-hybrid (Y2H), and affinity capture demonstrated in vivo interaction between Scm3Sp and Cnp1. GST-pulldowns in vitro showed the interaction to be direct (Pidoux et al., 2009). Microscopic localization of GFPtagged Scm3Sp revealed it localizes at centromeres, and chromatin immunoprecipitation (ChIP) showed Scm3Sp to be present in central domain chromatin. Critically, Cnp1 fails to localize at centromeres in scm3 mutants, which also exhibit severe chromosome segregation defects. Equally persuasive are results arguing that Scm3Sp is not a constituent of Cnp1 nucleosomes. Both groups made the striking finding that, whereas Cnp1 is present at the centromere continuously throughout the cell cycle, Scm3Sp is lost from centromeres at mitosis onset—when one might assume the integrity of Cnp1 nucleosomes to be most critical—and returns in late anaphase. Also, Scm3Sp centromere localization is retained in cnp1-1 mutants; thus, Scm3Sp localization is independent of Cnp1. That Cnp1 is released from chromatin by micrococcal nuclease treatment under conditions where Scm3Sp remains insoluble adds biochemical corroboration (Pidoux et al., 2009). This is not to say that Cnp1 nucleosomes are ‘‘normal.’’ ChIP experiments by both groups showed a relative lack of H2A and H2B associated with centromere central domain DNA. The H2A and H2B ChIP signals were similar to that of H3, correlating inversely with that of Cnp1. In scm3 mutants, where centromere enrichment of Cnp1 is lost, H2B and H3 occupancies increase, with H4 occupancy largely unchanged (Williams et al., 2009). The direct conclusion is that a majority of Cnp1 ‘‘nucleosomes’’
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 411
Molecular Cell
Previews
Figure 1. CenH3 Deposition in S. cerevisiae and S. pombe (A) Scm3Sc-bound Cse4 is localized to S. cerevisiae point centromeres through interaction with the Ndc10 subunit of the CDEIII-binding factor CBF3. There, it is assembled into an unconventional hexameric nucleosome (Mizuguchi et al., 2007). Scm3Sc displaces H2A-H2B from Cse4 histone octamers in vitro, but it is not known if this is the operative pathway in vivo. (B) The central domain of S. pombe regional centromeres consists of the central core (cnt) and innermost repeats (imr). The outer repeats (otr) are packaged into heterochromatin. Cnp1 nucleosomes, possibly lacking H2A and H2B, are present in central domain chromatin. Scm3Sp mediates the interaction between Cnp1 and the centromere-bound Mis16-Mis18 complex, which, along with Scm3Sp, dissociates from centromeres at metaphase and returns during late anaphase (Pidoux et al., 2009; Williams et al., 2009).
lack H2A-H2B dimers. Another possibility is that Cnp1 nucleosomes are dynamic. The underrepresentation of H2A-H2B dimers would reflect their ‘‘last on-first off’’ status
in the assembly/disassembly process, causing inefficient crosslinking. More work will be required for a definitive explanation, but the data are quite intriguing in light of
412 Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc.
recent reports of unconventional CenH3 nucleosomes in S. cerevisiae and D. melanogaster (Dalal et al., 2007; Mizuguchi et al., 2007). If Scm3Sp is a recruitment factor and not part of Cnp1 nucleosomes, how is Scm3Sp itself localized to centromeres? The Mis16-Mis18 complex is the most upstream factor required for Cnp1 deposition (Hayashi et al., 2004). The cell-cycledependent localization of Scm3Sp mirrors that of Mis18 (Fujita et al., 2007), and both Pidoux et al. (2009) and Williams et al. (2009) find that Scm3Sp is mislocalized in mis16 and mis18 mutants. Using GST pulldown, Pidoux et al. (2009) demonstrated a direct protein-protein interaction between Scm3Sp and Mis18 (but not Mis16), whereas Williams et al. (2009) found a Y2H interaction between Scm3Sp and Mis16 (but not Mis18). Additional analysis is required to resolve the discrepancy, but the combined evidence strongly suggests that Scm3Sp links Mis16-Mis18 and Cnp1 through direct protein-protein interaction (Figure 1). Scm3Sp localization is also dependent upon Mis6 and Sim4 (Pidoux et al., 2009; Williams et al., 2009); however, unlike Scm3Sp, the Mis6-Sim4 complex is present at centromeres constitutively, and its role in maintaining Scm3Sp is not known. Also unknown is whether delocalization of Mis16-Mis18-Scm3Sp during M phase is a prerequisite for Cnp1 assembly in the subsequent cell cycle, although cycle-dependent localization of Mis18 is conserved. Human Mis18, which is required for de novo CENP-A deposition, associates only briefly with centromeres between late anaphase and G1 (Fujita et al., 2007). Scm3 homologs are not found in vertebrates, but it is probable that functional analogs exist. Likely candidates would be proteins associated with Mis18, perhaps Mis18BP (Fujita et al., 2007). The identification by Pidoux et al. (2009) and Williams et al. (2009) of Scm3Sp as the proximate factor in Cnp1 targeting, linking Cnp1 to centromere-localized Mis16Mis18, is a major advance. Left unanswered is how Mis16-Mis18 finds it way to centromere DNA. Effort must now be directed backward in the pathway, toward elucidating the mechanism of Mis16Mis18 localization and its cell-cycle regulation.
Molecular Cell
Previews REFERENCES Camahort, R., Li, B., Florens, L., Swanson, S.K., Washburn, M.P., and Gerton, J.L. (2007). Mol. Cell 26, 853–865. Dalal, Y., Wang, H., Lindsay, S., and Henikoff, S. (2007). PLoS Biol. 5, e218. 10.1371/journal.pbio. 0050218. Fujita, Y., Hayashi, T., Kiyomitsu, T., Toyoda, Y., Kokubu, A., Obuse, C., and Yanagida, M. (2007). Dev. Cell 12, 17–30.
Furuyama, S., and Biggins, S. (2007). Proc. Natl. Acad. Sci. USA 104, 14706–14711. Hayashi, T., Fujita, Y., Iwasaki, O., Adachi, Y., Takahashi, K., and Yanagida, M. (2004). Cell 118, 715–729. Mizuguchi, G., Xiao, H., Wisniewski, J., Smith, M.M., and Wu, C. (2007). Cell 129, 1153–1164. Pidoux, A.L., Richardson, W., and Allshire, R.C. (2003). J. Cell Biol. 161, 295–307.
Pidoux, A.L., Choi, E.S., Abbott, J.K.R., Liu, X., Kagansky, A., Castillo, A.G., Hamilton, G.L., Richardson, W., Rappsilber, J., He, X., and Allshire, R.C. (2009). Mol. Cell 33, 299–311.
Stoler, S., Rogers, K., Weitze, S., Morey, L., Fitzgerald-Hayes, M., and Baker, R.E. (2007). Proc. Natl. Acad. Sci. USA 104, 10571–10576.
Williams, J.S., Hayashi, T., Yanagida, M., and Russell, P. (2009). Mol. Cell 33, 287–298.
Closing the Feedback Loop: How Cells ‘‘Count’’ Telomere-Bound Proteins Neal F. Lue1,* 1Department of Microbiology and Immunology, W.R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.001
Telomere length homeostasis is thought to occur via a ‘‘protein-counting’’ mechanism whereby high numbers of telomere-bound proteins inhibit telomerase activity. In a recent issue of Molecular Cell, Hirano et al. (2009) delineate the molecular interactions that underlie the budding yeast protein-counting machinery. Telomeres are specialized nucleoprotein structures that maintain the integrity of eukaryotic chromosomal termini by protecting them from fusion and recombination and by promoting their replication (de Lange, 2005). In most organisms, telomeric DNA comprises short repetitive sequences that are rich in G residues on the 30 end-containing strand. These repeats are maintained by a ribonucleoprotein known as telomerase, which acts as an unusual reverse transcriptase (Autexier and Lue, 2006). Both telomerebinding proteins and telomerase are critical for the maintenance of telomere integrity through multiple cell divisions, which in turn is pivotal in supporting genome stability and promoting cellular life span. Not surprisingly, telomere lengths are regulated to within a defined size range in organisms ranging from yeast to man. The homeostatic system has been extensively analyzed and is best understood in the budding yeast Saccharomyces cerevisiae. Classic studies by Marcand et al. (1997) led to the proposal that telomere-
length regulation is achieved through a ‘‘protein-counting’’ mechanism whereby higher numbers of proteins bound by a longer telomere repeat tract ultimately inhibit telomerase activity at that particular telomere (Figure 1). The key elements of such a cis-acting inhibitory feedback loop are apparent from the outset: the factor being counted (the sensor), the factor or structure that transmits the count (the transducer), and the factor that elongates short telomeres (the effector, i.e., telomerase). Initially, the counted protein was thought to be Rap1, the major double strand telomere repeat binding factor. Subsequent studies by the Blackburn group suggested that Rif1 and Rif2, which are recruited to telomeres by a Rap1 C-terminal domain, can be ‘‘counted’’ by the cell in the absence of Rap1 (Levy and Blackburn, 2004). More recently, a series of elegant experiments by several labs pointed to the MRX (Mre11-Rad50-Xrs2) complex and Tel1 as plausible transducers of the system; these proteins are regulated by the Rif proteins, preferen-
tially localize to short telomeres, and positively promote telomerase action at telomeres (Bianchi and Shore, 2007; Hector et al., 2007; Sabourin et al., 2007; Tseng et al., 2006). Importantly, these studies identified a plausible set of interactions that relay the signal from the transducers to the effector. In essence, the MRX complex, once bound to short telomeres, enhances the localization of Tel1, which in turn phosphorylates Cdc13 and possibly other factors to promote telomerase recruitment and activity (Figure 1). What has been conspicuously missing, however, is a plausible molecular mechanism to relay the signals from any sensor (Rap1, Rif1, and Rif2) to the transducer (MRX and Tel1). This is where the recent study by Hirano et al. (2009) provides some satisfying answers. Using a system that induces a specific chromosome break adjacent to either telomere repeats or the bacterial Tet operator (TetO) repeats, Hirano et al. (2009) examined how tethering varying numbers of Rap1, Rif1, or Rif2 next to the DNA ends affects MRX and Tel1 localization.
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 413