Article Figures & Data
Figures
- Figure 1. The CLRC complex plays a dominant in silencing telomeric heterochromatin.
(A) A schematic diagram of the shelterin, CLRC, and SHREC complexes at telomeres. The Ccq1 subunit of shelterin facilitates recruitment of both the CLRC and the SHREC complexes to telomeres. (B) Effects of Ccq1 truncation mutants on the transcriptional silencing of his3+ reporter gene inserted adjacent to the telomeric region. Equal amounts of 10-fold dilution series of cultures were spotted on YES or Pombe Medium Glutamate supplemented with uracil, leucine, and adenine (PMG ULA) (-histidine) plates. (C) Effects of the fusion of Ccq11–500 and Ccq11–439 mutants with Clr3 or Clr4 on the transcriptional silencing of his3+. The strains were spotted on plates as in (B).
- Figure 2. Biochemical characterization of the Ccq1–Raf2 interaction.
(A) Domain organization of Ccq1. The domains and motifs within Ccq1 that mediate interactions with Est1 (Moser et al, 2011), Tpz1 associating domain (TAD) (Jun et al, 2013; Harland et al, 2014), Raf2-binding motif (RBM) and Clr3-binding motif (CBM) (Armstrong et al, 2018) are designated. (B) Multiple sequence alignment of Schizosaccharomyces pombe Ccq1RBM and its homologues. Conserved residues of Ccq1RBM are boxed and highlighted in red. Red stars denote residues important for the Ccq1–Raf2 interaction. (C) Identification of the domain of Ccq1 that mediates interaction with Raf2 by yeast two-hybrid (Y2H) analysis. (D) Y2H assay to screen mutations of Ccq1RBM that disrupt the Ccq1RBM–Raf2 interaction. (E) Effects of Ccq1 mutations on the interaction of full-length Ccq1 (Ccq1FL) with Raf2 were examined in Y2H assays. (C, D, E) In (C, D, E), Raf2 was fused to Gal4 DNA-binding domain, and WT and mutant Ccq1 were individually fused to Gal4 activation domain. Error bars in the graph represent mean ± SEM. (F) Co-IP analysis of the interaction of Myc-tagged Raf2 with Flag-tagged wild-type or mutant Ccq1. The levels of each protein in input and IP samples were analyzed by immunoblotting with the indicated antibodies.
Source data are available for this figure.
Source Data for Figure 2[LSA-2021-01106_SdataF2.pdf]
- Figure S1.
Effect of Raf2-binding deficient Ccq1 mutantions on Ccq1-Tpz1 and Ccq1-Clr3 interactions by Y2H assay. (A, B) Ccq1 mutations that disrupt the Ccq1–Raf2 interaction have no effect on Ccq1-Tpz1 (A) and Ccq1-Clr3 (B) Yeast two-hybrid interactions. Tpz1 or Clr3 were fused to the Gal4 DNA binding domain, and WT and mutant Ccq1 were individually fused to Gal4 activation domain. Data are averages of three independent β-galactosidase measurements normalized to the wild-type interaction, arbitrarily set to 100. Error bars in the graph represent mean ± SEM.
- Figure S2. Effects of disruption of the Ccq1-Raf2 interaction on telomerase recruitment and telomere maintenance.
(A) Ccq1L511R, Ccq1V516R, and Ccq1Y518R mutant proteins were expressed at the wild-type level. Flag-tagged WT and mutant Ccq1 proteins were ectopically expressed under the control of the native Ccq1 promoter. The expression levels of GAPDH are used as a control. (B) Co-IP of Ccq1 and TER1 in vivo. Data are represented as mean ± SEM from three independent experiments. (C) Southern blot analysis of telomere lengths of WT and Raf2-binding deficient Ccq1 mutant strains (ccq1L511R and ccq1V516R). Genomic DNAs were digested with EcoR I and subjected to Southern blot analysis with a telomere-specific probe. The non-telomeric control was used as a relative-mobility control. (D) Microscopic analysis of WT, ccq1L511R, and ccq1V516R cells grown in liquid YES culture. Red scale bar indicates 10 μm.
- Figure 3. The Ccq1-Raf2 interaction mediates heterochromatin formation and CLRC enrichment at telomeres.
(A) A schematic figure of telomeric and subtelomeric regions in fission yeast Schizosaccharomyces pombe. Positions on x axis represent distances from telomeric repeats (van Emden et al, 2019). (B, C, D) ChIP-qPCR analysis of Ccq1 (B), Raf2 (C), and Clr4 (D) in WT, ccq1L511R, and ccq1V516R strains. Recruitment to the internal act1+ locus serves as a control for ChIP specificity. (E) Effects of Raf2-binding deficient mutations of Ccq1 (Ccq1L511R and Ccq1V516R) on the transcriptional silencing of his3+ reporter gene inserted adjacent to the telomeric region. (F) RT-qPCR analysis of the transcription of TERRA in WT and ccq1L511R and ccq1V516R mutant strains, normalized to that of act1+. The transcription at the cen-dg region was used as a control. (G, H) ChIP-qPCR analysis of H3K9me2 (G) and Swi6 (H) in WT and ccq1L511R cells. Recruitment to the internal act1+ locus serves as a control for ChIP specificity. (B, C, D, F, G, H) Data information: In (B, C, D, F, G, H), data are represented as means from three independent experiments. (B) In (B), the error bars represent mean ± SEM. *0.05 > P > 0.01 (t test).
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01106_SdataF3.xlsx]
- Figure 4. Ccq1-CLRC-mediated heterochromatin promotes nucleosome stability and shelterin enrichment at subtelomeres.
(A) ChIP-qPCR analysis of histone H3 in WT and ccq1L511R cells. Recruitment to the internal act1+ locus serves as a control for ChIP specificity. (B) Fold change of WT over ccq1L511R for H3K9me2 and H3 levels. (C, D, E, F) Effects of the Ccq1L511R mutation on telomere association for Taz1 (C), Rap1 (D), Poz1 (E) and Tpz1 (F) were measured by ChIP-qPCR assays. Recruitment to the internal act1+ locus serves as a control for ChIP specificity. (A, C, D, E, F) Data information: In (A, C, D, E, F), data are represented as means from three independent experiments. (A) In (A), the error bars represent mean ± SEM. *0.05 > P > 0.01; **P < 0.01 (t test).
Source data are available for this figure.
Source Data for Figure 4[LSA-2021-01106_SdataF4.xlsx]
- Figure 5. A schematic model for the nucleation and spreading of heterochromatin at subtelomeres by the shelterin and CLRC complexes.
The Ccq1-Raf2 interaction contributes to CLRC association with telomeric repeats and subtelomeric regions that catalyzes the histone H3K9 methylation, and the methylated histone H3K9 sequentially recruit Swi6 to initiate heterochromatin formation. Moreover, The CLRC association promotes nucleosome stability at subtelomeres, which further facilitates the methylation of histone H3K9 by CLRC. Finally, shelterin-mediated CLRC recruitment in turn facilitates the shelterin association with subtelomeric chromatin, and this positive feedback loop between the shelterin and the CLRC complexes plays a critical role in the nucleation and spreading of heterochromatin at subtelomeres.
Supplementary Materials
Table S1 Yeast strains used for this study.
Table S2 Oligonucleotides used in this study.
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