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The tail-module of yeast Mediator complex is required for telomere heterochromatin maintenance - PubMed

The tail-module of yeast Mediator complex is required for telomere heterochromatin maintenance

Jing Peng et al. Nucleic Acids Res. 2012 Jan.

Abstract

Eukaryotic chromosome ends have a DNA-protein complex structure termed telomere. Integrity of telomeres is essential for cell proliferation. Genome-wide screenings for telomere length maintenance genes identified several components of the transcriptional regulator, the Mediator complex. Our work provides evidence that Mediator is involved in telomere length regulation and telomere heterochromatin maintenance. Tail module of Mediator is required for telomere silencing by promoting or stabilizing Sir protein binding and spreading on telomeres. Mediator binds on telomere and may be a component of telomeric chromatin. Our study reveals a specific role of Mediator complex at the heterochromatic telomere and this function is specific to telomeres as it has no effect on the HMR locus.

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Figures

Figure 1.
Figure 1.

Mediator is involved in telomere length regulation. (A) The telomere length of all 15 viable Mediator mutants was examined by telomere Southern blot. The telomere lengths of the tested strains were quantified and the changes of telomere length compared with wild-type strain were indicated at the bottom of the corresponding lanes. (B) Telomere length of double mutants of different modules was monitored as in (A). (C) Expression of genes encoding telomerase components in Mediator mutants was detected by qRT-PCR. Statistical analyses in this study were calculated using Student's t-test (*P < 0.05 and **P < 0.01). (D) Telomere length analysis of the Mediator mutants overexpressing TLC1 with the CEN plasmid pRS316-TLC1. V: vector (pRS316), T: Tlc1 (pRS316-TLC1). The telomere length changes indicated at the bottom denote the differences between the mean values of two clones with or without TLC1 overexpression respectively.

Figure 2.
Figure 2.

Mediator tail module is required for telomere position effect and acts through Sir2. (A) Telomere position effect (TPE) of 14 Mediator mutants. Cells were grown in YPD and diluted to the same concentration. Five-fold serial dilutions of each strain were spotted to YC plates with or without 0.15% 5-FOA as indicated. (B) The expression of telomeric URA3 (TEL::URA3) reporter gene detected by qRT-PCR, and normalized to that of ACT1 gene. (C) TPE of Mediator single mutants and double mutants of Mediator and SIR2. (D) qRT-PCR results of expression of double mutants of Mediator and SIR2.

Figure 3.
Figure 3.

H4K16 acetylation increases and Sir2 binding is reduced at modified telomere VIIL in Mediator tail mutants. (A) Specificity of anti-H4K16Ac antibody was verified. Chromatin of wild-type and H4K16R mutant cells was extracted and subjected to western blot. (B) Schematic diagram of the modified telomere VIIL (m7L) and the locations of primers used in ChIP assay. (C) ChIP results of H4K16 acetylation level on telomere m7L. The wild-type value at the most distal locus (m7L-1) is arbitrarily set to 1 where the acetylation level is supposed to be the lowest. The statistical significances of difference in H4K16 acetylation between mutants and wild-type cells are provided (*P < 0.05 and **P < 0.01). (D) H4 level is detected by ChIP assay. The wild-type value at m7L-1 is set to 1. (E) Binding of 13Myc-tagged Sir2 on m7L was detected by ChIP assay using anti-Myc antibody.

Figure 4.
Figure 4.

Sir2 binding on native telomeres is decreased. (A–C) ChIP assays detecting Sir2 binding on native telomeres VIIL (n7L) (A), VIR (6R) (B) and IIIR (3R) (C). Primers mapped to each telomere are indicated on the top schematic diagrams.

Figure 5.
Figure 5.

Mediator is associated with both modified and natural telomeres independently of Sir2 or Rap1. (A) Binding of 13Myc-tagged Mediator subunits on telomere m7L. The LEU1 locus serves as a negative control where little Mediator binds (48). (B) Binding of Mediator on native telomeres 3R and 6R. (C) Binding of Mediator on telomeres m7L, 3R and 6R in sir2Δ strains. (D) Telomeric binding of Mediator subunits Med2 and Med3 in RAP1 or rap1-2 strains at 23°C or 37°C were detected by ChIP assay. (E–G) ChIP results of Mediator binding in wild-type, med20Δ or med15Δ cells on telomeres m7L (E), 3R (F) and 6R (G).

Figure 6.
Figure 6.

Mediator does not affect heterochromatin at the HMR locus. (A) Expression of endogenous HMRa1 gene was examined. (B) Mediator does not affect expression of HMRa1 in sir2Δ strains. (C) Schematic diagram of HMR region. Primers for ChIP assay were indicated. (D) Sir2 binding at HMR region in Mediator mutants.

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