Genetics and Genomics

Elimination of subtelomeric repeat sequences exerts little effect on telomere functions in Saccharomyces cerevisiae

Can Hu
Xue-Ting Zhu
Ming-Hong He
Yangyang Shao
Zhongjun Qin
Zhi-Jing Wu author has email address
Jin-Qiu Zhou author has email address

The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, 200031, China;
Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, 200032, China;
Key Laboratory of Systems Health Science of Zhejiang Province, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China

https://doi.org/10.7554/eLife.91223.2

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Figures and data

Telomere structures in SYn tlc1Δ survivors. Telomere Southern blotting assay was performed to examine telomere structure. The genomic DNA extracted from BY4742 (wild type) and SYn strains (labeled on top) was digested with XhoI and subjected to Southern hybridization with a TG1-3 probe. (A) Telomerase-proficient strains (labeled on top), whose chromosome numbers are labeled at the bottom. Two independent clones of each strain were examined. (B-D) SYn tlc1Δ survivors generated on plates. Four (BY4742 tlc1Δ) and fifteen (SYn tlc1Δ) individual survivor clones (labeled on top of each panel) of each strain were examined. “+” at the bottom indicates Type I survivors. “Δ” marks the survivors which are non-canonical Type I or Type II.

Survivor formation in SY12 tlc1Δ strain. (A) Schematic representation of chromosome (and telomere) structures (not drawn to scale) in the SY12 strain (left panel) and the Type X survivor (right panel). The Roman numerals, native chromosomes; the Arabic numerals on the left, chromosome numbers of SY12; yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres; black circles, centromere; vertical arrows and numbers, positions and lengths of the terminal Xhol digestion fragments detected by the telomeric TG1-3 probe. Chromosome numbers are omitted in the Type X survivor (right panel). (B) Cell viability assay in liquid medium. The growth of SY12 (labeled in black) and SY12 tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (C) Telomeric Southern blotting assay of SY12 tlc1Δ survivors.
Genomic DNAs prepared from SY12 tlc1Δ survivors assayed in (B) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (D) Telomere Southern blotting assay of SY12 tlc1Δ survivors obtained on solid medium. Genomic DNA from fifty independent SY12 tlc1Δ clones (labeled on top) was digested with XhoI and hybridized to a telomere-specific TG1–3 probe. Type II survivors: in orange; Type I survivors: in red; circular survivors: in blue; Type X survivors: in green; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12 strain are indicated on the left. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC). (E) Southern blotting results of an SY12 tlc1Δ Type II survivor at the 20^th re-streaks after TLC1 reintroduction. LC: loading control. (F) Schematic of three circular chromosomes and fusion sequences in the SY12 tlc1Δ-C1 survivor. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres. (G) Telomere Southern blotting analysis of an SY12 tlc1Δ circular survivor at the 20^th re-streak after TLC1 reintroduction. LC: loading control. (H) Telomere Southern blotting analysis of an SY12 tlc1Δ Type X survivor at the 20^th re-streak after TLC1 reintroduction. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. LC: loading control.

Characterization of SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ strains. (A) Schematic of chromosome structures in the SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ strains. Yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres. Vertical arrows and numbers indicate the positions and sizes of the sites and length of Xhol and PaeI-digested terminal fragments. (B) PCR analyses of the engineered sites of the individual telomeres (labeled on left) in BY4742, SY12, SY12^YΔ, SY12^YΔ+1XΔ, SY12^YΔ+2XΔ, SY12^YΔ+3XΔ, SY12^YΔ+4XΔ, SY12^XYΔ and SY12^XYΔ+Υ strains (labeled on top). Primer sequences for the PCR analyses are listed in supplementary file 1. RAP1 was an internal control. (C) Morphology of BY4742, SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ cells in the exponential growth phase (30°C in YPD). Shown are DIC images. Scale bar, 2 μm. (D) Growth analysis of the SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ strains. Several clones of the SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ strains were re-streaked on YPD plates 61 times at intervals of two days. Shown were the 3^rd, 31^st and 61^st re-streaks. (E) Growth analysis of BY4742, SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ cells in liquid culture. Error bars represent standard deviation (s.d.), n = 3. (F) FACS analysis of DNA content of BY4742, SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ cells. (G) Dotting assays on YPD plates at low (24°C) and high (37°C) temperatures, or on YPD plates containing methyl methane sulfonate (MMS), camptothecin (CPT) or hydroxyurea (HU) at the indicated concentrations. The BY4742 mre11Δ haploid strain serves as a negative control because Mre11 is involved in the repair of double-stranded breaks (Lewis et al., 2004).

Telomere length and telomere silencing analyses of SY12^YΔ, SY12^ΧΥΔ and SY12^XYΔ+Υ strains. (A) Southern blotting analysis of telomere length in SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ (labeled on top) cells. Genomic DNA prepared from three independent clones of SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ strains were digested with XhoI and PaeI, and then subjected to Southern blotting with a TG1-3 probe. The numbers in brackets indicate the telomere length of the corresponding chromosomes. (B) Expressions of MPH3 and HSP32 in ΒΥ4742, SY12, SY12^YΔ, SY12^XYΔ and SY12^XYΔ+Υ cells were detected by qRT-PCR. The numbers above the schematic line (lower panels) indicate the distance to the corresponding subtelomeric elements or telomeres. The RNA levels of MPH3 and HSP32 were normalized by ACT1. The wild-type value is arbitrarily set to 1. Error bars represent standard deviation (s.d.), n = 3. ‘ns’, p> 0.5 (Student’s t-test); *, P < 0.05 (Student’s t-test); **, P < 0.01 (Student’s t-test).; ****, P < 0.0001 (Student’s t-test).

Survivor analysis of SY12^YΔ tlc1Δ strain. (A) Cell viability assay in liquid medium. The growth of SY12^YΔ (labeled in black) and SY12^YΔ tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (B) Telomeric Southern blotting assay of SY12^YΔ tlc1Δ survivors. Genomic DNAs prepared from SY12^YΔ tlc1Δ survivors assayed in (A) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (C) Telomere Southern blotting analysis of SY12^YΔ tlc1Δ survivors obtained on solid medium. Genomic DNAs of fifty independent survivors (labeled 1 to 50 on top) were digested with XhoI and hybridized by a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12^YΔ strain are indicated on left. LC: loading control. (D, F) Southern blotting results of an SY12^YΔ tlc1Δ circular survivor (D) or an SY12^YΔ tlc1Δ Type II survivor (F) at the 20^th re-streaks after TLC1 reintroduction. LC: loading control. (E) Schematic of three circular chromosomes and fusion sequences in the SY12^YΔ tlc1Δ-C1 survivor. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired, dashes are deleted. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres.

Survivor analysis of SY12^XYΔ tlc1Δ and SY12^XYΔ+Y tlc1Δ strains. (A) Telomere Southern blotting analysis of SY12^XYΔ+Y tlc1Δ survivors obtained on solid medium. fifty independent survivors (labeled 1 to 50 on top) were randomly picked, and their genomic DNAs were digested with XhoI and subjected to the Southern blotting assay with a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. The sizes of individual telomere restriction fragments of the SY12^XYΔ+Y strain are indicated on the left. LC: loading control. (B) Telomeric Southern blotting assay of SY12^XYΔ tlc1Δ survivors obtained on solid medium. fifty independent survivors (labeled 1 to 50 on top) were randomly picked, and their genomic DNAs were digested with XhoI and subjected to the Southern blotting assay with a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. The sizes of individual telomere restriction fragments of the SY12^XYΔ+Y strain are indicated on the left. LC: loading control. (C) and (E) Schematic of three circular chromosomes and fusion sequences in the SY12^XYΔ+Y tlc1Δ-C1 and SY12^XYΔ tlc1Δ-C1 survivors, respectively. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired, dashes are deleted. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres. (D) Southern blotting results of an SY12^XYΔ+Y tlc1Δ circular survivor and an SY12 ^XYΔ+Y tlc1Δ Type II survivor at the 20^th re-streaks after TLC1 reintroduction. LC: loading control. (F) Southern blotting result of an SY12^XYΔ tlc1Δ Type II survivor at the 20^th re-streaks after TLC1 reintroduction. LC: loading control.

Characterization of SY12 strain. (A) Schematic representation of chromosome (and telomere) structures (not drawn to scale) in the SY12 strain. The Roman numerals, native chromosomes; the Arabic numerals on the left, chromosome numbers of SY12; yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres; black circles, centromere; vertical arrows and numbers, positions and lengths of the terminal Ndel digestion fragments detected by the telomeric TG1-3 probe. (B) Southern blotting analysis of telomere length in BY4742 and SY12 (labeled on top) cells. Genomic DNA prepared from two independent clones of BY4742 and SY12 strains was digested with NdeI, and then subjected to Southern blotting with a TG1-3 probe. The numbers in brackets indicate the telomere length of the corresponding chromosomes. The blot was then stripped and reprobed with a Y’ probe. The asterisk indicates the non-specific band.

Y’-elements have been eroded in SY12 tlc1Δ survivors. The blot in Figure 2D was then stripped and reprobed with a Y’ probe.

Borders of erosion of the SY12 tlc1Δ-C1 survivor are defined by PCR mapping. (A) Upper panel, a schematic diagram of the subtelomeric region of 0-60 kb proximal to I-L telomere is shown. Primer pairs (No. 1^L to 17^L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top; solid circles mean positive PCR products and open circles mean no PCR products with corresponding primer pairs. (B) Upper panel, a schematic diagram of the subtelomeric region of 0-50 kb proximal to X-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1^R to 42^R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (C) Upper panel, a schematic diagram of the subtelomeric region of 0-10 kb proximal to XIII-L telomeric TG1-3 sequence is shown. Primer pairs (No. 1^L to 6^L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (D) Upper panel, a schematic diagram of the subtelomeric region of 0-65 kb proximal to XI-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1^R to 25^R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (E) Upper panel, a schematic diagram of the subtelomeric region of 0-35 kb proximal to XVI-L telomeric TG1-3 sequence is shown. Primer pairs (No. 1^L to 29^L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (F) Upper panel, a schematic diagram of the subtelomeric region of 0-60 kb proximal to XIV-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1^R to 21^R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top.

PFGE result of circular survivors. Chromosomal DNA analysis of “circular survivors” SY12 tlc1Δ-C1, SY12^YΔ tlc1Δ-C1, SY12^XYΔ tlc1Δ-C1, SY12^XYΔ+Y tlc1Δ-C1 by PFGE. The S. cerevisiae strain SY15 (with a single circular chromosome) was used as control. As marker, the size of three chromosomes in wild type S. pombe strain is indicated on right.

Telomere structure determination of Type X survivor. Schematic representation of chromosome (and telomere) structures (not drawn to scale) in the Type X survivor. The Roman numerals, native chromosomes; the Arabic numerals on the left, chromosome numbers of SY12; yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres; black circles, centromere. The sequence in yellow belongs to X-element. Bases in rad are mis-paired. The dotted line in green represents the sequence of Y’-element is lost.

Survivor formation in SY12 tlc1Δ rad52Δ strain. (A) Cell viability assay in liquid culture. The growth of SY12 (labeled in black), SY12 tlc1Δ (two clones labeled in purple and blue respectively) and SY12 tlc1Δ rad52Δ (three clones labeled in violet, pink and carmine respectively) were monitored every 24 hr for 12 days. (B) Telomere Southern blotting assay of SY12 tlc1Δ rad52Δ survivors obtained on solid medium. Genomic DNA from twenty-five independent clones (labeled on top) was digested with XhoI and hybridized to a telomere-specific TG1–3 probe. Circular survivors: in blue; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12 strain are indicated on the left. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC).

Southern blotting result of SY12 tlc1Δ rad51Δ and SY12 tlc1Δ rad50Δ survivors. Genomic DNAs were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. Type II survivors: in orange; Type I survivors: in red; Circular survivors: in blue; Type X survivors: in green; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12 strain are indicated on the left. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC).

Schematics of CRISPR–Cas9-mediated deletion of X-and Y’-elements on individual chromosomes in SY12 cells. The specific DNA sequences centromere-proximal to the subtelomeric region (site S1) were cleaved by the Cas9 nuclease with the guidance of gRNA1. Homologous recombination between the broken ends and the provided donors led to the deletion of X-and Y’-elements. Galactose induction of gRNA2 on pCas9 caused the cleavage at site S2 and the URA3 marker was counter-selected on 5’-FOA plates. Deletion of X-and Y’-elements were determined by PCR analysis with a primer located within the deletion region and another upstream of the region (indicated by purple arrows).

PCR mapping of the borders of erosion in SY12^YΔ tlc1Δ-C1 cell. Red lines indicate the regions which have been deleted in the SY12^YΔ strain.

Survivor formation in SY12 ^YΔ tlc1Δ rad52Δ strain. (A) Cell viability assay in liquid culture. The growth of SY12 ^YΔ (labeled in black), SY12 ^YΔ tlc1Δ (two clones labeled in purple and blue respectively) and SY12 ^YΔ tlc1Δ rad52Δ (three clones labeled in violet, pink and carmine respectively) strains were monitored every 24 hr for 12 days. (B) Telomere Southern blotting assay of SY12 ^YΔ tlc1Δ rad52Δ survivors obtained on solid medium. Genomic DNA from twenty-five independent clones (labeled on top) was digested with XhoI and hybridized to a telomere-specific TG1–3 probe.
Circular survivors: in blue; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12 ^YΔ strain are indicated on the left. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC).

SY12^XYΔ+Y tlc1Δ and SY12^XYΔ tlc1Δ strains form Type II survivors in liquid culture. (A) Cell viability assay in liquid medium: The growth of SY12^XYΔ+Y (labeled in black) and SY12^XYΔ+Y tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (B) Telomeric Southern blotting assay of SY12 ^XYΔ+Y tlc1Δ survivors. Genomic DNAs prepared from survivors in (A) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (C) Cell viability assay in liquid medium: The growth of SY12^XYΔ (labeled in black) and SY12^XYΔ tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (D) Telomeric Southern blotting assay of SY12 ^XYΔ tlc1Δ survivors. Genomic DNAs prepared from survivors in (C) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe.

PCR mapping of the borders of erosion in SY12^XYΔ+Υ tlc1Δ-C1 cell. Red lines indicate the regions which are absent in the SY12^XYΔ+Υ strain.

PCR mapping of the borders of erosion in SY12^XYΔ tlc1Δ-C1 cell. Red lines indicate the regions which have been deleted in the SY12^XYΔ strain.

Survivor formation in SY12 ^XYΔ+Y tlc1Δ rad52Δ and SY12 ^XYΔ tlc1Δ rad52Δ strains. (A) Cell viability assay in liquid culture. The growth of SY12 ^XYΔ+Y (labeled in black), SY12 ^XYΔ+Y tlc1Δ (two clones labeled in purple and blue respectively) and SY12 ^XYΔ+Y tlc1Δ rad52Δ (three clones labeled in violet, pink and carmine respectively) strains shown in the left panel; the survivor formation in SY12 ^XYΔ (labeled in black), SY12 ^XYΔ tlc1Δ (two clones labeled in purple and blue respectively) and SY12 ^XYΔ tlc1Δ rad52Δ (three clones labeled in violet, pink and carmine respectively) strains shown in the right panel. They were monitored every 24 hr for 12 days. (B) Telomere Southern blotting assay of SY12 ^XYΔ+Y tlc1Δ rad52Δ (upper) and SY12 ^XYΔ tlc1Δ rad52Δ (under) survivors obtained on solid medium. Genomic DNA from twenty-five independent clones (labeled on top) was digested with XhoI and hybridized to a telomere-specific TG1–3 probe. Circular survivors: in blue; atypical survivors: in black. Theoretical telomere restriction fragments of each strain are indicated on the left. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC).

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