Stable inheritance of telomere chromatin structure and function in the absence of telomeric repeats - PubMed (original) (raw)

. 2003 Sep 15;17(18):2271-82.

doi: 10.1101/gad.1112103. Epub 2003 Sep 2.

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Stable inheritance of telomere chromatin structure and function in the absence of telomeric repeats

Mahito Sadaie et al. Genes Dev. 2003.

Abstract

It is generally believed that telomeric repeats are a necessary and sufficient cis-element for telomere function. Here we show that telomere structure and meiotic function are stably inherited in fission yeast circular chromosomes that have lost all telomeric repeats. We found that the telomeric repeat binding protein, Taz1, and the heterochromatin protein, Swi6, remain associated with subtelomeres in the absence of telomeric repeats. We also found that the fusion point of circular chromosomes that lack telomeric repeats associates with SPB (the yeast counterpart of the centrosome) in the premeiotic horsetail stage, similarly to wild-type telomeres. However, a taz1+ deletion/reintroduction experiment revealed that the maintenance of Taz1 binding and premeiotic function is achieved via different strategies. Taz1 is recruited to subtelomeres by an autonomous element present in subtelomeric DNA, thus in a genetic mechanism. In contrast, the premeiotic subtelomere-SPB association is maintained in an epigenetic manner. These results shed light on the previously unrecognized role played by the subtelomere and underscore the robust nature of the functional telomere complex that is maintained by both genetic and epigenetic mechanisms. Furthermore, we suggest that the establishment and the maintenance of the functional telomere complex are mechanistically distinguishable.

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Figures

Figure 1.

Figure 1.

Subtelomeric regions of circular chromosomes behave like endogenous telomeres in the horsetail stage. (A) Cos212-SPB association in the horsetail stage of derivative cells. Cells in the horsetail stage were examined simultaneously by FISH analysis using the cos212 probe, which detects subtelomeric DNA of Chromosomes I and II (cos212), and by IF analysis using anti-Sad1 antibodies that detect SPB. Cos212-reactive sequences are present at the four ends of Chromosomes I and II, but absent on Chromosome III (lower panel). (B) Summary of cos212-SPB association frequencies in eight independent derivative clones (a-h) as well as in wild-type cells. Cos212-SPB associations were classified in terms of FISH results according to the criteria shown below. (C,D) The fusion points of circular Chromosome III do not cluster at the SPB in the horsetail stage in Types A, B, and AB clones (clones a, b, and g, respectively). The subtelomeric regions of Chromosome III (rDNA) were detected by FISH analysis using the rDNA probe (lower panel). rDNA-SPB associations were classified in terms of FISH results according to the criteria shown below. (E,F) Efficiencies of homolog pairing of circular chromosomes in Types A, B, and AB clones (clones a, b, and g, respectively). The cosmid clone that detects Chromosome I (cos25G10), II (cos839), or III (cos1322) was individually used as a FISH probe. The percentages of cells in which FISH signals appeared as a single dot or two closely located dots are shown.

Figure 2.

Figure 2.

Larger regions of TAS are lost in Type B clones than in Type A clones. (A) A composite physical map of pNSU70, pNSU56, pB15E1, and cos212, which are overlapping clones containing fission yeast telomere-associated sequences (TASs). The locations of the probes used in Southern hybridization are indicated by thick bars, together with the distance (in kilobases) of the probe from the telomeric repeats (parentheses). Restriction sites relevant to the experiment are shown. (B) Relative copy numbers of 562-hybridizing sequences in derivative clones. _Cla_I-digested genomic DNA was simultaneously hybridized with the 562 probe and the his1 probe. The calculated relative intensities of the 562 band to the his1 band are shown at the bottom. (C) Genomic DNAs digested with indicated restriction enzymes were hybridized with the E11, E12, E13, or E14 probe. Ethidium-bromide-stained DNAs are shown as a loading control (right). In wild-type cells, doublets were detected with probes E11 and E12. The upper band is lost in all derivative clones, and may be derived from the Chromosome III subtelomere. (D) Schematic representation of subtelomeric deletion in Types A, AB, and B clones.

Figure 3.

Figure 3.

Taz1 interacts with TAS in both wild-type and Type A derivative clones. (A) Approximate and relative positions of the PCR products used to detect Taz1-TAS interaction in B and C. The results shown in B and C are summarized below the map. (B) Results of the ChIP assay for Taz1-HA and TAS interactions. As control, wild-type cells in which Taz1 was not tagged (HA tag -) were used in addition to wild-type cells expressing HA-tagged Taz1 (HA tag +). Whole-cell extracts (W) were analyzed to monitor extraction efficiencies. The K region, located between mat2 and mat3 loci, was used as a negative control. (C) Fragments 562 and E11-E14 were analyzed as in B using Type A cells (clone a) expressing HA-tagged or HA-untagged Taz1.

Figure 4.

Figure 4.

Taz1-dependent epigenetic inheritance of subtelomere-SPB association in Type A clones. (A) Cos212-SPB association is disrupted in the Δ_taz1_ Type A clone and is not restored by the reintroduction of taz1+. Cos212-SPB association frequencies of indicated strains in the Δ_taz1_ background are shown as in Figure 1B. taz1+-HA (re) indicates Taz1-HA-expressing strain in which taz1+-HA was integrated at the taz1+ locus of each Δ_taz1_ strain. (B) Typical images of Type A cells (clone a) with or without taz1+ in the horsetail stage. (C) Taz1 localizes at the leading tip of the horsetail nuclei in wild-type and Type A cells, whereas it dissociates from DNA in Type B cells. Taz1-HA and SPB were simultaneously stained with anti-HA antibodies and anti-Sad1 antibodies, respectively. (Top) Wild type. (Middle) Type A clone. (Bottom) Type B clone. (D) The Taz1-subtelomere association is restored by the reintroduction of taz1+ into Δ_taz1_ Type A clones. ChIP experiments were conducted as described in Figure 3. taz1+-HA indicates strains in which the endogenous taz1+ was tagged by HA, and taz1+-HA (re) indicates Δ_taz1_ strains in which taz1+-HA was integrated to the original taz1+ locus.

Figure 5.

Figure 5.

(A) Swi6 is present at the fusion points of circular chromosomes. The subtelomeric DNAs of Chromosomes I and II (cos212) were detected with cos212 FISH, and the frequencies of all cos212 signals overlapping with Swi6-GFP signals were calculated for wild-type, Type A (clone a), and Type B (clone b) cells. (B) Taz1 is dispensable for Swi6 localization at telomeres. Frequencies of Swi6-GFP-cos212 colocalization in wild-type cells (having linear chromosomes) and Δ_taz1_ (originated from the wild-type) cells are shown as in A.

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