A mechanism for telomere-specific telomere length regulation

Abstract

Telomeric DNA, composed of short, direct repeats, is of crucial importance for chromosome stability. Due to intrinsic problems with replicating this DNA, the repeat tracts shorten at each cell division. Once repeat tracts become critically short, a telomeric stress signal induces cellular senescence and division arrest, which eventually may lead to devastating age-related degenerative diseases associated with dysfunctional telomers. Conversely, maintenance of telomere length by telomerase upregulation is a hallmark of cancer. Therefore, telomere length is a critical determinant of telomere function. How telomere length is established and molecular mechanisms for telomere-specific length regulation remained unknown. Here we show that subtelomeric chromatin is a determinant for how telomere equilibrium set-length is established in cis. The results demonstrate that telomerase recruitment mediated by the telomere-associated Sir4 protein is modulated on chromosome 3L in a telomere-specific way. Increased Sir4 abundance on subtelomeric heterochromatin of this specific telomere leads to telomere lengthening of only that telomere in cis, but not at other telomeres. Therefore, this work describes a mechanism for a how telomere-specific repeat tract length can be established. Further, our results will force the evaluation of telomere length away from a generalized view to a more telomere-specific consideration.

Keywords: Ku-proteins; Telomerase RNA; Telomerase recruitment; Telomere length maintenance; Telomeric chromatin.

Extended Figure 1.

a. Southern blot for telomeric fragments and plotted telomeric tract lengths on DNA derived from three clones each of W3749A WT, sir1Δ (GTY11), sir2Δ (GTY12), sir3Δ (GTY13) and sir4Δ (EPY031). DNA was digested with XhoI and hybridized with a telomeric repeat fragment. TEL06R and Y’ tract lengths are indicated on right of blot. Quantitative statistics: ns for not significant and *** for p < 0.001, t-test. b. Southern blot with DNA derived from three clones of W3749A, four clones of rad52Δ MATa, two clones of rad52Δ MATα and one clone of W3749α. DNA was digested with PvuII and probed with a telomeric repeat fragment. TEL03L, TEL11L and TEL06R telomeric repeat tract lengths are indicated on right. (ns for difference not significant, t-test). c. 1. Table with TeloPCR results from Fig. 1a. Mean ± SD and p-value are detailed for TEL03L, TEL06R and TEL11R. 2. Table with Southern blot results from Fig. 1b. Mean ± SD and p-value are detailed for TEL03L, TEL11L and TEL06R. 3. Table with southern blot results from Extended Fig. 1a. Mean ± SD and p-value are detailed for TEL06R and Y’-telomeres. 4. Table with Southern blot results from Extended Fig. 1b. Mean ± SD and p-value are detailed for TEL03L, TEL11L, and TEL06R.

Extended Figure 1.

a. Southern blot for telomeric fragments and plotted telomeric tract lengths on DNA derived from three clones each of W3749A WT, sir1Δ (GTY11), sir2Δ (GTY12), sir3Δ (GTY13) and sir4Δ (EPY031). DNA was digested with XhoI and hybridized with a telomeric repeat fragment. TEL06R and Y’ tract lengths are indicated on right of blot. Quantitative statistics: ns for not significant and *** for p < 0.001, t-test. b. Southern blot with DNA derived from three clones of W3749A, four clones of rad52Δ MATa, two clones of rad52Δ MATα and one clone of W3749α. DNA was digested with PvuII and probed with a telomeric repeat fragment. TEL03L, TEL11L and TEL06R telomeric repeat tract lengths are indicated on right. (ns for difference not significant, t-test). c. 1. Table with TeloPCR results from Fig. 1a. Mean ± SD and p-value are detailed for TEL03L, TEL06R and TEL11R. 2. Table with Southern blot results from Fig. 1b. Mean ± SD and p-value are detailed for TEL03L, TEL11L and TEL06R. 3. Table with southern blot results from Extended Fig. 1a. Mean ± SD and p-value are detailed for TEL06R and Y’-telomeres. 4. Table with Southern blot results from Extended Fig. 1b. Mean ± SD and p-value are detailed for TEL03L, TEL11L, and TEL06R.

Extended Figure 2.

a. 1 Table with initial telomeric tract lengths from Fig. 2a. Mean ± SD and p-value are detailed for TEL03L, TEL11R, TEL06R and Y’-telomeres. 2. All values for the loss rate analyses from Fig. 2d. Mean ± SD and p-value are detailed for TEL03L, TEL11R, TEL06R and Y’-telomeres. b. Schematic drawing of the construction of strains GTY26 and GTY27. GTY26 was constructed with a DNA fragment containing a LEU2 gene flanked by RS sites, the TEL03L X-element and a short seed of telomeric repeat. GTY27 was constructed with a DNA fragment containing a LEU2 gene flanked by RS sites, the TEL01L X-element and a short seed of telomeric repeat. After loss of the LEU2 marker gene by recombination, cells were grown for 100 generations. The positions of the HindIII restriction sites on TEL03L in strains GTY26, GTY27 and the WT are indicated.

Extended Figure 3.

a. 1. Table with Southern blot results from Fig. 3a. Mean ± SD and p-value are detailed for Tel03L. 2. Table with Southern blot results from Fig. 3c. Mean ± SD and p-value are detailed for TEL03L. b. TeloPCR of 3 clones of TEL03L-03L (GTY26) and 3 clones of TEL03L-1L (GTY27). Telomeric DNA fragments amplified from TEL03L. c. Southern blot of 3 clones of W3749A WT, 3 clones of hmlΔ (GTY19), 3 clones of hmrΔ (GTY20) digested with PvuII and probed with a telomeric repeat fragment. d. Southern blot of 3 clones of W3749A WT, 3 clones of hmlΔ (GTY19), 3 clones of hmrΔ (GTY20) digested with XhoI and probed with a telomeric repeat fragment.

Extended Figure 4.

a. Table with Southern blot results from Fig. 4a. Mean ± SD and p-value are detailed for TEL03L, TEL11L, and TEL06R. b. Nanopore sequencing mapping of telomere length distributions of TEL03L and TEL06R in BY4741 WT versus BY4741 sir3Δ cells. Straight line indicates overall telomere length average of all telomeres and the dashed line is the individual telomere length average of that particular telomere.

Extended Figure 5.

a. Southern blot of DNA from W3749A WT cells, cells with the tlc1Δ48 allele (GTY10), cells with the hmlΔ allele (GTY02) and cells with the sir4Δ allele (EPY031). DNA was digested with PvuII and probed with a TEL03L unique probe. b. Left: Southern blot with the same DNA as in Fig. 5a but digested with XhoI and probed with a telomeric repeat fragment. Right: DNA of cells of the indicated genotypes as in Fig. 5a but digested with XhoI and probed with a telomeric repeat fragment. c. All telomeric repeat tract quantifications with calculated means of at least three independent clones each, standard deviations, and significance calculations for the gels in Fig. 5 and Extended Fig. 5.

Extended Figure 5.

a. Southern blot of DNA from W3749A WT cells, cells with the tlc1Δ48 allele (GTY10), cells with the hmlΔ allele (GTY02) and cells with the sir4Δ allele (EPY031). DNA was digested with PvuII and probed with a TEL03L unique probe. b. Left: Southern blot with the same DNA as in Fig. 5a but digested with XhoI and probed with a telomeric repeat fragment. Right: DNA of cells of the indicated genotypes as in Fig. 5a but digested with XhoI and probed with a telomeric repeat fragment. c. All telomeric repeat tract quantifications with calculated means of at least three independent clones each, standard deviations, and significance calculations for the gels in Fig. 5 and Extended Fig. 5.

Extended Figure 6.

a. Detail of the single amino acid mutation in tbf1–453 allele. b. Growth characteristics of cells with the tbf1–453 allele vs WT. The four strains were derived from a microdissected heterozygous diploid strain. c. Selected results of mRNA seq analysis of total RNA derived from WT and tbf1–453 cells. log2 ratios of the indicated genes are listed (See Data Availability section for access to all data). (+) indicates that the promoter of that gene contains a predicted Tbf1 binding site; (−) indicates that this gene does not contain such a site. d. Quantitative ChIP-PCR for Sir4 binding in telomere proximal areas of the indicated telomeres. Results from the procedure with untagged Sir4 or on the MET3 locus served as negative controls (−); the HMRa locus (+) serves as a positive control. Primers are on the indicated telomeres at a distance from the X-element as indicated in brackets in kb. The differences between TBF1 WT and tbf1–453 on the indicated loci are significant with ** p < 0.01; t-test. For the other loci (not indicated) p < 0.05; t-test.

Extended Figure 7

a. Southern blot of DNA from W3749A WT cells, cells with the rpd3Δ allele (GTY31), cells with the tbf1–453 allele (ELY268–6b), and cells with the sir4Δ allele (EPY031). The DNA was digested with XhoI (left part), or PvuII (right part) and hybridized to a telomeric repeat fragment. b. The quantified telomeric tract lengths of TEL03L, TEL11L and TEL06R vs the indicated genotypes are plotted (* for p < 0.05, ** for p < 0.01 and *** for p < 0.001, t-test). c. All telomeric repeat tract quantifications with calculated means of at least three independent clones each, standard deviations, and significance calculations for the gels in Fig. 6 and Extended Fig. 7.

Extended Figure 7

a. Southern blot of DNA from W3749A WT cells, cells with the rpd3Δ allele (GTY31), cells with the tbf1–453 allele (ELY268–6b), and cells with the sir4Δ allele (EPY031). The DNA was digested with XhoI (left part), or PvuII (right part) and hybridized to a telomeric repeat fragment. b. The quantified telomeric tract lengths of TEL03L, TEL11L and TEL06R vs the indicated genotypes are plotted (* for p < 0.05, ** for p < 0.01 and *** for p < 0.001, t-test). c. All telomeric repeat tract quantifications with calculated means of at least three independent clones each, standard deviations, and significance calculations for the gels in Fig. 6 and Extended Fig. 7.

Figure 1.. Exceptionally long telomere set length on TEL03L

a TeloPCR analysis with DNA of 6 clones of WT W3749A. Telomeric DNA fragments amplified from TEL03L, TEL06R, and TEL11R. b. Quantified telomeric tract lengths of the TeloPCR results in a (TEL03L, TEL06R and TEL11R; *** for p < 0.001, t-test). c Southern blot of DNA from 3 clones of WT W3749A digested with PvuII, hybridized with a radiolabeled telomeric repeat fragment. Telomeric bands TEL03L, TEL11L and TEL06R were sized and calculated tract lengths are indicated on right. d. Telomere length distribution of each individual chromosome end from a BY4741 WT strain as obtained by Oxford Nanopore Sequencing. Straight vertical line indicates overall telomeric repeat average and the dashed line is the individual telomeric repeat length average of that particular telomere. Data for TEL04R is missing due to insufficient number of reads for this telomere.

Figure 2.. Comparable telomere shortening rates on all telomeres

a. Initial telomeric tract lengths for 3 clones of Est3-FRB are shown for TEL03L, TEL06R, TEL11R and Y’-telomeres. b. Changing telomeric tract lengths of TEL11R, TEL06R and Y’-telomeres during culture outgrowth (14 days or indicated population doublings) of Est3-FRB cells. c. Same as b but for TEL03L. d. Calculated telomeric repeat loss rates (in bp/population doublings; bp/PDs) for Est1-FRB and Est3-FRB strains are tabled for TEL03L, TEL11R, TEL06R and Y’-telomeres. (ns for difference not significant, t-test).

Figure 3.

a. Southern blot and telomeric tract lengths of Tel03L-03L cells (GTY26), Tel03L-1L cells (GTY27) and WT cells with DNA digested with HindIII and probed with a TEL03L unique probe. b. Quantified telomeric tract lengths (TEL03L) of the blot shown in a. (ns for difference not significant, t-test). c. Southern blot of DNA derived from W3749A WT cells, cells harboring hmlΔ (GTY19), and cells harboring hmrΔ (GTY20). DNA was digested with PvuII and probed with a TEL03L unique probe. d. Quantified telomeric tract lengths (TEL03L) of the blot shown in c. (* for p < 0.05 and ns for difference not significant, t-test).

Figure 4:. Long repeat tracts on TEL03L depend on the presence of Sir-proteins.

a. Southern blot of DNA derived from W3749A WT cells, sir1Δ (GTY11), of sir2Δ (GTY12), sir3Δ (GTY13) and sir4Δ (EPY031) cells, digested with PvuII and probed with a telomeric repeat fragment. b. Quantified telomeric tract lengths from a of TEL03L, TEL11L and TEL06R are plotted with respect of the genotype (ns for difference not significant, * for p < 0.05 and *** for p < 0.001, t-test).

Figure 5.. Long repeat tracts on TEL03L are mediated by increased telomerase recruitment.

a. Left: Southern blot of DNA derived from W3749A WT cells, cells harboring the tlc1Δ48 allele (GTY10), cells harboring the hmlΔ allele (GTY02), or cells with a sir4Δ allele (EPY031). DNA was digested with PvuII and hybridized to a telomeric repeat fragment. Right: Southern blot as on left, but with W3749A WT cells, cells with a yku80Δ allele (EPY129), EPY129 cells in which the yku80Δ allele is complemented by a plasmid-borne WT YKU80 (indicated as pYKU80; strain GTY23) and EPY129 cells in which the yku80Δ allele is complemented by a plasmid-borne yku80-L140A allele (indicated as pyku80-L140A; strain GTY24). Bands labeled with an * are new recombinant X-telomere bands, but not TEL03L (see Extended Fig. 5a). b. Quantified telomeric tract lengths of TEL03L, TEL11L, TEL06R, and Y’-telomeres are plotted on the graph vs the indicated genotypes (ns for not significant, * for p < 0.05, t-test). Southern blots for the data are in Fig. 5a, Extended Fig. 5a, b)

Figure 6.. A boundary element mutation causes Sir4 spreading and telomere elongation.

a Southern blot of DNA from W3749A WT cells, cells with the tbf1–453 allele (ELY268–6b), cells with both the tbf1– 453 and sir2Δ alleles (CLY05), cells with both the tbf1–453 and sir3Δ alleles (CLY06), cells with both the tbf1–453 and sir4Δ alleles (GTY28). DNAs were digested with PvuII and probed with a telomeric repeat fragment. b. The telomeric tract lengths of TEL03L, TEL11L andTEL06R from the gel in a were quantified and plotted on the graph vs the indicated genotype (ns for not significant, * for p < 0.05 and ** for p < 0.01, t-test).