From the ¶ Fondazione Istituto Pasteur-Fondazione
Cenci-Bolognetti, c/o Dipartimento di Genetica e Biologia Molecolare,
Università La Sapienza, P.le Aldo Moro 5, 00185 Roma, Italy and
Centro Acidi Nucleici, Consiglio Nazionale delle Ricerche,
Roma Italy
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ABSTRACT |
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We have defined the in vivo heterochromatin structure of the left telomere of Saccharomyces cerevisiae chromosome III (LIII). Analysis of heterochromatin of a single telomere was so far lacking, due to the difficulties intrinsic to the highly repetitive nature of telomeric sequences. In LIII, the terminal (C1-3A)n repetitive sequences are followed by a complete X element and by the single copy Ty5-1 retrotransposon. Both the telosome and the X element exhibit overall resistance to micrococcal nuclease digestion reflecting their tight chromatin structure organization. The X element contains protein complexes and irregularly distributed but well localized nucleosomes. In contrast, a regular array of phased nucleosomes is associated with the promoter region of Ty5-1 and with the more centromere-proximal sequences. The lack of a structural component of yeast telomeres, the SIR3 protein, does not alter the overall tight organization of the X element but causes a nucleosome rearrangement within the promoter region of Ty5-1 and releases Ty5-1 silencing. Thus, Sir3p links the modification of the heterochromatin structure with loss of transcriptional silencing.
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INTRODUCTION |
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Telomeres play an essential role in cell biology in stabilizing chromosomes and facilitating complete replication of chromosomal termini. Telomeric DNA usually contains tandem repetitions of a short motif flanked by subtelomeric middle repetitive sequences (1). In yeast, telomeric sequences are composed of about 350 base pairs containing the (C1-3A)n repeats and are followed by two main subtelomeric sequences: the Y' and X elements (2). Y elements are highly conserved and are found in about 70% of the telomeres (2-5). X elements are present in all telomeres and can exist in two main forms: a complete form containing the X core and the STR-A,B,C,D (6) elements or a short form containing essentially the X core or part of it (2, 4-6). The complete X is found in about 80% of the telomeres, whereas uncomplete forms are found in the remaining 20%.
Previous reports have referred to the chromatin structure of yeast telomeres as heterochromatin. This denomination is based on structural and functional similarities that yeast telomeres share with Drosophila heterochromatin (7-9). In Saccharomyces cerevisiae the terminal (C1-3A)n repeats are organized into a nuclease-resistant structure called telosome (10, 11) that does not contain nucleosomes and is associated with the protein RAP1 (10-13). This protein binds to the repetitive (C1-3A)n sequences (14, 15) and interacts with other proteins including RIF1, RIF2, SIR3, and SIR4 (16-18). The proteins SIR3 and SIR4 interact with each other, RAP1, and the amino terminus of the histones H3 and H4. Thus, the building of the telomeric heterochromatic structures in yeast involves complex homotypic and heterotypic interactions.
Although the chromatin structure of yeast telomeres is probably the best known among all eukaryotes, its specific organization is still poorly understood. It is known that both Y' and X elements contain nucleosomes (10). However, their distribution within both elements has not been described previously.
A previous report has described that the Ty5-1 retrotransposon, a subtelomeric transcriptional unit located in S. cerevisiae LIII, undergoes telomeric silencing (19). This retrotransposon is silenced in wild-type strains but is derepressed in a sir3 mutant. We describe here the chromatin structure of LIII and its influence on the silencing of Ty5-1. A link between Ty5-1 silencing and a specific Sir3p-dependent heterochromatin structure is established.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains and Culture Conditions-- S. cerevisiae wild-type strains LJY153 and PKY501 and mutant LJY155 (bearing deletion of the SIR3 gene) (sir3) (32, 33) were grown at 28 °C on SC medium containing glucose to an A600 of about 1.0/ml.
Nuclease Sensitivity Analyses-- Cells from 50-ml cultures were collected by centrifugation, treated with zymolyase, and digested with micrococcal nuclease (MNase)1 or Dnase I as described previously (34). For digestions with DNase I, the incubation buffer was supplemented with 6 mM MgCl2. After digestions of chromatin or naked DNA, the sensitivity to the enzymes was analyzed by the indirect end-labeling technique (20). The DNA samples were purified, digested with BamHI, resolved in agarose gels, and transferred to nylon membranes. The cutting profiles generated by both enzymes were visualized after hybridization with an internal probe from Ty5-1. This probe abuts the BamHI site selected for the analyses and extends from position 1495 to 1725 of S. cerevisiae chromosome (35). Nucleosomal spacing analyses were performed with MNase. After digestion of chromatin with MNase, the purified DNA samples were directly resolved in agarose gels generating a nucleosome ladder that could be visualized by staining with ethidium bromide. The DNA samples were then transferred to nylon membranes and hybridized with the probes as indicated in the legend to Fig. 2.
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RESULTS |
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The Chromatin Organization of the STR, X-core, and Ty5-1 Sequences-- The chromatin structure of S. cerevisiae LIII has been analyzed by using the enzymes MNase and DNase I. Permeabilized cells were digested with increasing concentrations of MNase and the sensitivity to the enzyme analyzed by indirect end-labeling (20) of purified DNA using an internal probe from Ty5-1 (Fig. 1a). Fig. 1b shows the MNase digestion profiles of chromatin from two different wild-type strains (PKY501 and LJY153) and of naked DNA. One fuzzy telomeric band is observed in the lanes containing undigested naked DNA or chromatin from PKY501 (Fig. 1b, band a). In contrast, two bands are observed in the lane containing undigested chromatin from LJY153 (Fig. 1b, bands b and c). These bands correspond to two different subpopulations of telomeric fragments containing different average amounts of (C1-3A)n repeats.2
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The Heterochromatin Structure in the Absence of Sir3p-- The chromatin structure of S. cerevisiae LIII has been also analyzed in a sir3 mutant (LJY155). Fig. 3 shows the MNase and DNase I digestion profiles of the mutant and of its isogenic wild-type strain (LJY153). As in the wild-type strain, the X element shows overall protection against MNase in the sir3 mutant (Fig. 3a). Thus, SIR3 is not necessary to keep the closed overall organization of the X element. In addition, the digestion pattern of the X element is quite similar in both strains suggesting that the lack of Sir3p does not cause major changes in the specific chromatin structure of the X element.
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DISCUSSION |
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The URA3 reporter gene and other reporter genes placed immediately adjacent to terminal (C1-3A)n repeats in the absence of subtelomeric sequences are silenced (22). Therefore, the special heterochromatic organization of the X elements is not required for the spreading of transcriptional telomeric silencing.
Yeast telomeres repress the expression of adjacent genes (7). This repression, referred to as telomere position effect, requires the integrity of telomeric proteins like RAP1, SIR2, SIR3, SIR4, and the amino termini of histones H3 and H4 (23-25). SIR3 and SIR4 interact among them, with RAP1 and with the amino terminus of the core histones. Thus, they have been proposed to physically connect telosomes and the nucleosomes located in the telomeric regions that undergo silencing (17, 26-28). Genetic and biochemical data support this notion. SIR3 and SIR4 require RAP1 and the amino termini of histones H3 and H4 to silence telomeric genes (23-25). SIR3, SIR4, and RAP1 localize by immunofluorescence to a number of foci near the nuclear periphery, being the amino termini of histones H3 and H4 required for the perinuclear localization of SIR3 and SIR4. The positioning of these foci inside the nuclei is coincident with hybridization signals of subtelomeric repeats (26, 29, 30). In addition, SIR3, SIR4, RAP1, and histone proteins coimmunoprecipitate and are found at the same distance from the telomeres (27, 31).
We have shown in this report that the lack of Sir3p affects the chromatin structure of the X element and of the adjacent Ty5-1 retrotransposon in LIII. These results provide evidence for in vivo interactions between Sir3p and nucleosomes in the X element and in the Ty5-1 retrotransposon.
Ty5-1 has been previously described to be silenced in its natural context in a SIR3 dependent manner (19). The promoter region of the retrotransposon is located at about 1.3 kilobases from the end of LIII. This result is in agreement with previous studies showing that Sir3p can be immunolocalized up to 2-3 kilobases from the right telomere of S. cerevisiae chromosome VI (27, 31). Since the chromatin structure of the Ty5-1 promoter region is altered in a sir3 mutant, Sir3p establishes a link between chromatin remodeling and transcriptional telomeric silencing. The SIR complex could function as a stapler joining nucleosomes from the X element and from the Ty5-1 retrotransposon and impair the access of the transcriptional machinery to the Ty5-1 promoter.
In wild-type strains, the 5' region of Ty5-1 shows overall sensitivity to MNase similar to the sensitivity of bulk DNA (data not shown) and contains a normal array of nucleosomes. Thus, this heterochromatic region does not show specific structural features. However, according to previous results (27), the 5' region of Ty5-1 should associate with proteins like SIR2, SIR3, or SIR4. Therefore, these proteins should interact among them and with the amino termini of histones H3 and H4 without affecting the accessibility of MNase to the linker internucleosomal regions.
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ACKNOWLEDGEMENT |
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The support of G. P. Ateneo, La Sapienza is acknowledged. We are indebted to M. Grunstein for encouragement and the generous gift of all yeast strains used in this study.
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FOOTNOTES |
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* This work was supported by European Community Human Capital Mobility CHRX-CT94-0047.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Instituto de Bioquímica Vegetal y
Fotosíntesis (Universidad de Sevilla-CSIC), Centro de
Investigaciones Isla de la Cartuja, c/Américo Vespucio s/n, 41092 Sevilla, Spain.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 39-6-4991-2880; Fax: 39-6-4440-812; E-mail: Dimauro{at}axrma.uniroma1.it.
1 The abbreviation used is: MNase, micrococcal nuclease.
2 M. A. Vega-Palas, S. Venditti, and E. Di Mauro, unpublished observations.
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REFERENCES |
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