Repressive and non-repressive chromatin at native telomeres in Saccharomyces cerevisiae

Abstract

Background: In Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression.

Results: Chromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure.

Conclusion: Our data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression.

Figure 1

Comparison of chromatin structures by indirect-end-label analysis at three telomeres of Saccharoyces cerevisiae. (A) Schematic of a URA3-yEGFP marked native telomere showing relevant restriction sites. Probes used for indirect end-labelling are indicated by arrows. (B) The subtelomeric chromatin structure of the truncated VIIL and native IIIR and XIL telomeres of S. cerevisiae was analysed by MNase digestion and indirect end labelling with the indicated probe. The chromatin structure of an isogenic strain containing the URA3-yEGFP construct at the native URA3 locus was also analysed. A control MNase digest of deproteinized DNA (D), and marker bands generated by digestion with StuI and PstI (M) are also shown. The position of the inserted URA3-yEGFP cassette is shown by a light grey box with hatching to indicate the URA3-yEGFP CDS; the TATA box (T) is indicated with a black bar. Restriction sites are numbered from the URA3 start codon. Three promoter-associated hypersensitive sites are indicated by black arrow heads and an array of evenly spaced hypersensitive sites, present at the VIIL and XIL telomeres, by white arrow heads. Inferred nucleosome positions are shown by ovals. The most telomere-proximal open reading frames, YGL256W (e), YKL224C (f), and YCR107W (g) are indicated by arrows to the left of each blot. (C) The chromatin structure downstream of the URA3-yEGFP reporter was detected using a probe on the telomere proximal side of the StuI site. Marker bands were obtained by digestion with StuI and XmaI. MNase hypersensitive sites adjacent to the core X binding sites are indicated by grey arrows. A schematic of the IIIR and XIL telomeres is shown with the core X ACS and Abf1 binding sites indicated by black bars. The truncation end VIIL is identical except that it lacks the core X and STR repeats.

Figure 2

The Sir complex is required for the formation of repressive chromatin at a native telomere. (A) Frequency of fluoroorotic acid (FOA) resistance in isogenic Δsir::KanMX strains containing the URA3-yEGFP marker adjacent to the core X element at the indicated telomere. The mean and standard deviation of FOA resistance is given for each strain. (B) Chromatin structures of the XIL and IIIR telomeres were analysed in Δsir1::KanMX, Δsir2::KanMX, Δsir3::KanMX and Δsir4::KanMX strains, by MNase digestion and indirect end labelling, as described for Figure 1B.

Figure 3

Chromatin alterations towards the XIL centromere in a yku80 strain. (A) Frequency of fluoroorotic acid (FOA) resistance in Δyku80::KanMX strains containing the URA3-yEGFP marker adjacent to the core X element at the indicated telomere. The mean and standard deviation of FOA resistance is given for each strain. (B) The subtelomeric chromatin structure of the XIL telomere was analysed in the Δyku80::KanMX strain, by MNase digestion and indirect end labelling, using a probe adjacent to the BstXI site within yEGFP. The marker (M) was generated by digestion of purified DNA with BstXI and PstI. An MNase hypersensitive site within the reporter open reading frame is indicated by an asterisk, other labelling is as for Figure 1B.

Figure 4

Chromatin alterations at the XIL telomere in core X mutants. (A) Frequency of fluoroorotic acid (FOA) resistance in strains containing the URA3 marker adjacent to the core X element at the XIL telomere. The core X^mut and core X^mut, Δyku80::KanMX strains contain mutations of the ARS consensus sequence (ACS) and Abf1 binding sites at the XIL core X element. The mean and standard deviation of FOA resistance is given for each strain. (B & C) Chromatin structure analysis of the XIL subtelomere in the core X^mut and core X^mut, Δyku80::KanMX strains was performed as described for Figure 1B and 1C. A schematic of the XIL telomere is shown, with black bars indicating the core X ACS and Abf1 binding sites. MNase hypersensitive sites that are altered in the core X^mut strain are indicated by grey arrows.

Figure 5

Histone modifiers are required for silencing but not nucleosome positioning at telomeres. (A) Frequency of fluoroorotic acid (FOA) resistance in isogenic Δbre1::KanMX, Δdot1::KanMX, Δset1::KanMX, Δsas2::KanMX, and Δbdf1::KanMX strains containing the URA3-yEGFP marker adjacent to the core X element at the indicated telomere. The mean and standard deviation of FOA resistance is given for each strain. (B) The subtelomeric chromatin structure of the XIL telomere was analysed in the Δbre1::KanMX, Δdot1::KanMX, and Δbdf1::KanMX strains, by MNase digestion and indirect end labelling, as for Figure 1B.

Figure 6

Model of repressive and non-repressive chromatin at Saccharomyces cerevisiae telomeres. Schematic shows a URA3 marked telomere with the positions of the URA3 coding sequence, upstream activating sequence (UAS), and TATA box (T) indicated. (A) The non-repressive state is characterized by an open promoter conformation and the presence of unpositioned nucleosomes (light grey circles) upstream of the URA3 promoter region. This structure permits transcription (grey arrow) of the URA3 gene. (B) The repressive state is formed in two steps: (1) Phased nucleosomes are positioned upstream of the URA3 gene by a mechanism dependent on the Sir proteins (2 - 4). Interaction of the Sir proteins with this upstream region could occur by looping or folding-back of the telomere (not depicted). (2) Full repression is established by an additional step, such as further Sir recruitment or histone modification, which results in the URA3 promoter assuming a closed chromatin structure.