Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins
2004; Springer Nature; Volume: 23; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7600144
ISSN1460-2075
AutoresAngela Taddei, Florence Hediger, Frank Neumann, Christoph Bauer, Susan M. Gasser,
Tópico(s)Plant Gene Expression Analysis
ResumoArticle11 March 2004free access Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins Angela Taddei Angela Taddei Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Florence Hediger Florence Hediger Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Frank R Neumann Frank R Neumann Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Christoph Bauer Christoph Bauer NCCR Frontiers in Genetics, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland NCCR Frontiers in Genetics, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Angela Taddei Angela Taddei Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Florence Hediger Florence Hediger Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Frank R Neumann Frank R Neumann Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Christoph Bauer Christoph Bauer NCCR Frontiers in Genetics, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Susan M Gasser Corresponding Author Susan M Gasser Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland NCCR Frontiers in Genetics, Quai Ernest-Ansermet, Geneva, Switzerland Search for more papers by this author Author Information Angela Taddei1, Florence Hediger1, Frank R Neumann1, Christoph Bauer2 and Susan M Gasser 1,2 1Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet, Geneva, Switzerland 2NCCR Frontiers in Genetics, Quai Ernest-Ansermet, Geneva, Switzerland *Corresponding author. Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland. Tel.: +41 22 379 6127; Fax: +41 22 379 6868; E-mail: [email protected] The EMBO Journal (2004)23:1301-1312https://doi.org/10.1038/sj.emboj.7600144 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4PAD) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4PAD-mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku- and Esc1-dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope. Introduction Chromosomes assume a nonrandom distribution in interphase nuclei (Marshall, 2002). The function of their spatial arrangement is largely unknown, although recent evidence suggests that subnuclear compartments contribute to the establishment and maintenance of epigenetic controls over eukaryotic gene expression (Fisher and Merkenschlager, 2002). In yeast, flies and man, the protective ends of chromosomes known as telomeres nucleate the formation of an altered chromatin structure that represses the transcription of adjacent RNA pol II genes in a heritable fashion (termed telomere position effect or TPE; reviewed in Huang, 2002; Perrod and Gasser, 2003). Repressed telomeric chromatin in yeast is found near the nuclear envelope (NE) in discrete foci that are far fewer than the number of chromosomal ends. These clusters of telomeric DNA colocalize with pools of silent information regulatory proteins (Sir2–4; Gotta et al, 1996), which are essential for TPE. Intriguingly, a similar spatial juxtaposition of telomeres can be observed in the parasite Plasmodia, where the clustering favors subtelomeric gene conversion events between virulence factor loci (Freitas-Junior et al, 2000). In yeast, DNA elements known as silencers bind the sequence-specific factors Abf1, Rap1 and ORC, which nucleate the binding of the SIR complex at the cryptic mating-type loci, HML and HMR (Rusche et al, 2003). At telomeres, on the other hand, Rap1 binds the terminal [TG1–3]n repeat and cooperates with the end-binding complex YKu70/YKu80 to recruit SIR proteins (Moretti et al, 1994; Cockell et al, 1995). Once bound, the Sir complex spreads along adjacent nucleosomes preferentially binding underacetylated histone tails (Renauld et al, 1993; Hecht et al, 1996; Rusche et al, 2002). Several observations support the proposal that subnuclear organization influences silencing. First, the delocalization of SIR factors from telomeres promotes repression at silencer-flanked loci that usually remain active due to their distance from chromosomal ends (Maillet et al, 1996, 2001; Marcand et al, 1996). This suggests that telomeric foci sequester silencing factors from nontelomeric sites of action. Second, the repression of HML and other silencer-flanked reporters is aided by their proximity to telomeres (Thompson et al, 1994; Maillet et al, 1996; Marcand et al, 1996). Third, silencing can be restored to a derepressed allele of HMR by targeting proteins of the endoplasmic reticulum to the locus via a Gal4 DNA-binding domain (e.g. Yif1, Yip1; Andrulis et al, 1998). These membrane-spanning hybrids accumulate in NE and are thought to promote repression by tethering the reporter gene near telomeric pools of SIR proteins. It has never been demonstrated, however, that the Yif-targeted reporter genes are actually recruited to the NE. The proteins that mediate the perinuclear anchoring of yeast telomeres and silent loci are unknown, although mutations in YKU or SIR2–4 do affect telomere position. Indeed, the deletion of either subunit of the Ku heterodimer delocalizes roughly half of the yeast telomeres from the NE (Laroche et al, 1998), but leaves truncated telomeres tethered (Tham et al, 2001; Hediger et al, 2002a). The complete delocalization of certain telomeres, such as Tel 14L or a truncated Tel 6R (Tel 6Rt), was detected only after deletion of both sir4 and yku70, suggesting that redundant anchoring mechanisms exist (Hediger et al, 2002a). Because the restoration of silent chromatin allowed anchoring in the absence of Ku but in a Sir4-dependent manner, it was proposed that chromatin-bound SIR complexes and Ku define two partially redundant tethering pathways. Among the silencing factors in yeast, Sir4 is most likely to act as a chromatin anchor. First, ectopic expression of its coiled-coil carboxy-terminal fragment leads to the release of full-length Sir3 and Sir4 from an insoluble chromatin fraction (Cockell et al, 1995) and loss of TPE. Indeed, based on the homology between this domain and human lamins A and C (Diffley and Stillman, 1989), it has been speculated that Sir4 might substitute for the nuclear lamina, which yeast lack. Finally, a penultimate subdomain of Sir4 (aa 950–1262 called Sir4PAD for partitioning and anchoring domain) was found to confer efficient mitotic partitioning on otherwise unstable plasmids and to impair the free rotation of DNA (Ansari and Gartenberg, 1997). One mechanism that could account for both phenotypes would be the anchoring of DNA to a symmetrically segregating nuclear component. Recent work shows that plasmid partitioning by Sir4PAD requires Esc1 (establishes silent chromatin), a protein that associates with Sir4, imparts silencing when targeted to a crippled silencer-flanked reporter at HMR, and accumulates at the nuclear periphery when overexpressed (Andrulis et al, 2002). These data gave rise to the proposal that the Sir4–Esc1 interaction might link silent chromatin to a scaffolding at the nuclear periphery. Unfortunately, this hypothesis could not be tested in loss-of-function alleles, because Esc1, like Ku and Sir4, promotes and stabilizes silent chromatin. Indeed, to determine which elements mediate chromatin anchoring, it was necessary to separate the silencing functions of the major telomere-associated proteins from any tethering activity they might have. We show here that a silencing-incompetent YKu80 monomer and a subdomain of Sir4 can each relocalize chromatin to the nuclear periphery without nucleating silent chromatin. Furthermore, we show that Esc1 functions as a perinuclear anchor for this silencing-deficient domain of Sir4, but not as its exclusive binding site. Sir4PAD tethers chromatin through two pathways: one requiring yKu and the second Esc1. Finally, we show that these two pathways cooperate to anchor native telomeres in a cell-cycle-regulated manner. We propose that, unlike the chromatin components Sir4 and yKu, Esc1 plays a structural role determining chromosome domain position in vivo. Results An assay for chromatin relocalization We have developed a cytological screen for proteins that impart a specific nuclear localization to an otherwise randomly positioned chromosomal segment. Four LexA operators (lexAop) were linked to an array of 256 lacop-binding sites and integrated at an internal active locus >50 kb from either the centromere or the right telomere of chromosome 6, near ARS607 (called Chr6int; Figure 1A). When LacI–GFP is expressed in these cells, it binds the array creating a single focus of GFP fluorescence. The ability of various LexA fusion proteins to influence the subnuclear position of the array can then be assessed. Figure 1.An assay for perinuclear chromatin anchoring. (A) Strain GA-1461 bears lacop repeats and LexA-binding sites at an internal locus on the right arm of Chr 6 (Chr6int) and a GFP–Nup49 fusion. A typical single-plane confocal image superimposed on a phase image is shown. Chr6int can be mapped to one of three concentric zones of equal surface by using the ratio of the spot to pore measurement (black) over the nuclear diameter (red; see Materials ods). (B, C) LexA (B) or LexA–Yif1 (C) was expressed in GA-1461, and GFP spot positions were classified by cell-cycle stage. Bar graphs present the percentage of spots (y-axis; n=number of cells analyzed) per zone (x-axis). The dotted bar at 33% indicates a random distribution, and zone 1 distributions that are significantly different from random are indicated by * based on 95% confidence values (p). (D) Characteristic equatorial images of the GFP pore and Chr6int locus in GA-1461 expressing the indicated LexA fusion. In the insets, a 5 min track of Chr6int imaged by confocal microscopy at 1.5 s intervals is projected in red onto a single nuclear section (Heun et al, 2001b). Bar=1 μm. (E) LexA–Yif1 fusion was overexpressed in GA-1461 (WT) and in isogenic strains bearing complete yku70 or sir4 deletions. Here zone 1 values are compared. See complete statistics in Table I. Download figure Download PowerPoint Three-dimensional (3D) focal stacks were collected to measure the distance between LacI–GFP foci and the nuclear membrane (tagged with GFP–Nup49). Positions measured in several hundred cells show that the Chr6int lacop array is randomly distributed among three concentric nuclear zones of equal surface, with or without LexA (Figure 1B). Spot position is monitored in one focal plane with zones normalized to the measured nuclear diameter in this plane. To show that relocalization can occur, we first targeted LexA fused to an integral membrane protein called Yif1 (Andrulis et al, 1998) to the Chr6int locus. We observe a significant enrichment of Chr6int in the outermost nuclear zone throughout the cell cycle (Figure 1C). Given that the fluorescent GFP–Nup49 ring occupies a large fraction of zone 1, the presence of a chromosomal signal in this zone indicates close NE–DNA contact (<180 nm). We conclude that the LexA–Yif1 fusion, which promotes repression when targeted to a crippled silencer-flanked reporter gene (Andrulis et al, 1998), can indeed relocalize a chromosomal locus to the nuclear periphery. Because neither the silencing nor anchorage is 100% efficient, we monitored the residence time of the tagged locus at the periphery using time-lapse microscopy (Hediger et al, 2002a; Heun et al, 2001b). A total of 300–400 sequential images were collected at 1.5 s intervals from individual cells expressing either LexA or LexA–Yif1. Without LexA–Yif1, the locus moves freely in the nucleoplasm without any discernible perinuclear constraint (Figure 1D). In the presence of LexA–Yif1, the tagged locus oscillates back and forth along the NE, much like the movement of natural telomeres (Hediger et al, 2002a). Movements away from the nuclear periphery are occasional. We conclude that an internal nonsilenced chromosomal locus becomes significantly but reversibly associated with the NE by binding LexA–Yif1. Importantly, these results demonstrate that our chromatin 'relocation' assay can be used to identify protein domains that tether chromosomes to the nuclear periphery. Identification of silencing-defective YKU80 and SIR4 alleles Sir4 and the Ku complex represented logical anchoring candidates based on loss-of-function analyses. However, YKu80 and full-length Sir4, as well as its N- or C-terminal domains, nucleate silent chromatin when targeted to DNA (Marcand et al, 1996; Martin et al, 1999). Thus, in order to test a chromatin relocation activity in the absence of silent chromatin, we sought fusions of Sir4 and Ku that would separate silencing and anchoring activities. For Sir4, we fused LexA to the Sir4PAD domain, which unlike other Sir4 domains has no dominant-negative effect on TPE (Ansari and Gartenberg, 1997). For the Ku heterodimer, on the other hand, we created mutant forms of yku80 by degenerate PCR. Two alleles were identified (yku80-4 and yku80-9; see Figure 2A) that are unable to restore TPE in the yku80 null background. Figure 2.Silencing-incompetent yku80-4 and Sir4PAD anchor chromatin at the nuclear periphery. (A) Scheme of the different domains and mutants used in targeted silencing and relocation assays. Dashed boxes correspond to putative coiled-coil domains. (B) Different LexA fusions were expressed from pAT4 for a targeted silencing assay monitored in GA-2050 strain carrying TRP1 at HMR adjacent to a modified E silencer: E and B elements are replaced by four lexAop (designated Aeb). Serial 10-fold dilutions of GA-2050 expressing the indicated LexA fusions are grown on SC lacking leucine (−leu) or leucine and tryptophan (−leu −trp) to monitor the level of silencing (no growth on −trp). Sir2 overexpression is known to impair growth (Cockell et al, 2000). Efficiencies of TRP1 expression and of Chr6int relocalization (% zone 1; see Figure 3) are indicated on the right. (C–E) Two-hybrid interactions between the indicated fusion proteins were tested in GA-180 with appropriate bait and prey plasmids, and a lacZ reporter gene. Bait constructs of LexA–Sir4PAD (C) -Esc1C (D) -YKu70 or -YKu80 (E) fusions are constitutively expressed, while prey constructs (full-length Sir2p, subdomains of Sir3, Sir4 and Esc1) are galactose-inducible (pJG45–pGAL1). β-Galactosidase values are means of quadruplicate assay presented in arbitrary units. Download figure Download PowerPoint To test whether these proteins nucleate silencing when targeted to a crippled HM locus, we inserted a TRP1 reporter gene at HMR and replaced the Rap1 and Abf1 sites of the E silencer with four lexAop sites (creating a crippled silencer; c.f. Andrulis et al, 1998). Growth of serially diluted cultures in the presence or absence of tryptophan allows a qualitative measure of TRP1 repression. Figure 2B shows that whereas targeted LexA–yku80-9 confers robust silencing relative to LexA–Yif1 (100 × difference), no silencing was seen with LexA–yku80-4, LexA–Sir4PAD or LexA alone. LexA fused to either full-length Sir2 or the C-terminal 540 residues of Esc1 (Esc1c) also silenced efficiently, in agreement with previous reports (Cockell et al, 2000; Andrulis et al, 2002). It was important to show not only that the Sir4PAD domain and yku80-4 fail to promote repression, but that they also fail to bind other SIR components. By two-hybrid analysis we detect no significant interaction of Sir4PAD with any of the domains of Sir2, Sir3 and Sir4 that are known to be critical for repression (Figure 2C). On the other hand, we detect a strong interaction between Sir4PAD and Esc1C, as reported, when either protein is used as bait or prey (Andrulis et al, 2002; Figure 2C and D). We next tested the two yku80 mutant proteins for two-hybrid interaction with Sir4C, an interaction previously reported for YKu70 (Tsukamoto et al, 1997). The mutant that supports targeted silencing (yku80-9) binds Sir4C robustly, while the one deficient for targeted silencing (yku80-4) does not. Importantly, the yku80-9/Sir4C interaction does not require YKu70 (Figure 2E). Because LexA-yku80-4 and LexA–Sir4PAD neither promote targeted silencing nor bind the silencing relevant domains of SIR proteins, we are able to test their anchoring activity independently of Sir complex formation. Chromatin relocation activities of YKu80, Sir4PAD, Esc1 and Sir2 We expressed the LexA fusions discussed above in the tagged Chr6int strain to monitor their abilities to relocalize DNA to the NE (Figure 3). Quantitative analysis shows that both YKu80 fusions produce significant relocation of the GFP focus to the outermost zone of the nucleus. The tethering activity is observed in G1- and S-phase cells (zone 1 values ranging from 51%, p=10−10 to 61%, p=10−6), although not in G2-phase cells, where the locus remains random (24 to 29%, p=0.055). Statistical analyses are summarized in Table I. Importantly, the anchoring efficiencies of the two yku80 mutants are very similar, despite their opposite behavior with respect to silencing. This demonstrates the separation of function we sought to obtain (Figure 3A). Figure 3.YKu80, Sir4PAD and Esc1C relocalize chromatin to the nuclear periphery. The position of Chr6int with respect to the three concentric nuclear zones was determined as in Figure 1 in GA-1461 expressing the following fusions: (A) LexA–yku80-4 and LexA–yku80-9; (B) aa 960–1262 of Sir4 (Sir4PAD); (C) aa 1124–1658 of Esc1 (Esc1C). (D) LexA–Sir2 was expressed either in GA-1461 or its sir4 derivative (GA-1994). See Table I for statistics. * and the dotted bar are as in Figure 1. Download figure Download PowerPoint Table 1. Localization of lacop -tagged loci (n) and significance (p value) for zone 1 enrichment WT yku70 esc1 sir4 yku70 esc1 G1 phase Chr 6int+anchors LexA–Esc1C % zone 1 82 71 42 60 ND n;p 144;<1 × 10−16 78;3 × 10−12 83;0.1 172;2 × 10−13 LexA–yku70 % zone 1 49 ND ND ND ND n;p 349;1 × 10−9 LexA–yku80-4 % zone 1 51 37 50 47 ND n;p 296;1 × 10−10 130;0.4 240;8 × 10−7 274;3 × 10−6 LexA–yku80-9 % zone 1 53 ND ND ND ND n;p 296;2 × 10−5 LexA–Sir4PAD % zone 1 65 64 64 77 36 n;p 131;1 × 10−14 247;<1 × 10−16 182;<1 × 10−16 60;<1 × 10−12 140;0.6 LexA–Sir2 % zone 1 56 ND ND 33 ND n;p 172;1 × 10−10 240;0.8 LexA–Yif1 % zone 1 54 55 57 51 59 n;p 197;1 × 10−9 92;1 × 10−6 99;9 × 10−7 121;3 × 10−5 141;1 × 10−10 Telomeres Tel 14L % zone 1 53 42 50 36 40 n;p 257;3 × 10−11 198;7 × 10−3 291;2 × 10−9 292;0.34 361;1 × 10−2 Tel 6Rt % zone 1 52 47 41 43 38 n;p 431 0.05). (B) Scheme of Sir4PAD and YKu80 anchoring pathways. Download figure Download PowerPoint LexA–Sir4PAD chromatin relocation activity was unaffected by loss of either Esc1 or YKu70 individually (Figure 5A), yet we see a complete loss of the Sir4PAD tethering activity in the absence of both Esc1 and Ku, throughout the cell cycle. This result strongly suggests that Esc1 and Ku provide two parallel anchorage pathways for Sir4PAD (Figure 5B). Indeed, the Sir4–Esc1 interaction can account for the yKu-independent anchoring described previously (Tham et al, 2001; Hediger et al, 2002a). Intriguingly, LexA–Sir4PAD anchors more efficiently in the absence of endogenous Sir4. This latter could reflect competition between the full-length Sir4 and Sir4PAD for a limiting number of perinuclear binding sites. We next examined whether anchoring via Esc1C requires Sir4 or Ku. Consistent with Esc1 localization results (Figure 4B), the Esc1C-mediated relocation of Chr6int shows little dependence on either SIR4 or YKU70 (Figure 5C). It is sensitive, on the other hand, to the deletion of the genomic ESC1 gene. The simplest explanation for this is that Esc1 homodimerizes through its C-terminal domain, which is present in the LexA fusion, but requires its N-terminus for perinuclear localization. It is not clear why the requirement for full-length Esc1 is more pronounced in G1 phase. To rule out nonspecific effects of these mutations, we show that the anchoring activity of LexA–Yif1 promotes significant anchoring in all deletion strains tested (Figure 1E, Table I). The YKu80 anchoring activity requires YKu70 only in G1-phase cells We next examined whether the chromatin relocation activity associated with yku80-4 requires the Ku heterodimer. Surprisingly, we find that yku80-4-mediated anchoring is lost in the yku70 mutant in G1 phase, yet is maintained in this strain during S phase (Figure 5D). It appears, therefore, that there is one anchorage site in G1 for the Ku heterodimer, and another in S phase that recognizes YKu80 directly. Coupled with the fact that LexA–YKu70 can relocate chromatin to the periphery only in G1 phase (Table I), we propose that cell-cycle-regulated changes in the Ku complex influence anchoring. We further observe that the yku80-4 anchorage function is sensitive to deletion of ESC1 uniquely in S phase (Figure 5D). This suggests an S-phase-specific interaction between YKu80 and Esc1, or between YKu80 and an Esc1-dependent factor, distinct from the G1-phase Ku anchor (Figure 5D). In summary, YKu80-mediated anchoring in G1 phase requires YKu70 but is independent of Esc1, while in S-phase cells it requires Esc1 (Figure 5B). Importantly, these cell-cycle variations mirror differences observed for the positioning of GFP-tagged telomeres in yku70 mutants (Hediger et al, 2002a). Esc1 tethers natural telomeres and promotes repression Recent analysis of telomere localization in living cells has shown telomere-specific effect of YKU mutations: Tel 6R was randomly localized in a yku70 deletion strain, while Tel 14L and a truncated Tel 6Rt remained significantly perinuclear. Important
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