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. 2018 Jan 25;46(2):704-716.
doi: 10.1093/nar/gkx1223.

Fission yeast Ccq1 is a modulator of telomerase activity

Christine A Armstrong  1 , Vera Moiseeva  1 , Laura C Collopy  1 , Siân R Pearson  1 , Tomalika R Ullah  1   2 , Shidong T Xi  1   3 , Jennifer Martin  1   2 , Shaan Subramaniam  1   3 , Sara Marelli  1 , Hanna Amelina  1 , Kazunori Tomita  1
Affiliations

Fission yeast Ccq1 is a modulator of telomerase activity

Christine A Armstrong et al. Nucleic Acids Res. .

Abstract

Shelterin, the telomeric protein complex, plays a crucial role in telomere homeostasis. In fission yeast, telomerase is recruited to chromosome ends by the shelterin component Tpz1 and its binding partner Ccq1, where telomerase binds to the 3' overhang to add telomeric repeats. Recruitment is initiated by the interaction of Ccq1 with the telomerase subunit Est1. However, how telomerase is released following elongation remains to be established. Here, we show that Ccq1 also has a role in the suppression of telomere elongation, when coupled with the Clr4 histone H3 methyl-transferase complex and the Clr3 histone deacetylase and nucleosome remodelling complex, SHREC. We have dissected the functions of Ccq1 by establishing a Ccq1-Est1 fusion system, which bypasses the telomerase recruitment step. We demonstrate that Ccq1 forms two distinct complexes for positive and negative telomerase regulation, with Est1 and Clr3 respectively. The negative form of Ccq1 promotes dissociation of Ccq1-telomerase from Tpz1, thereby restricting local telomerase activity. The Clr4 complex also has a negative regulation activity with Ccq1, independently of SHREC. Thus, we propose a model in which Ccq1-Est1 recruits telomerase to mediate telomere extension, whilst elongated telomeric DNA recruits Ccq1 with the chromatin-remodelling complexes, which in turn releases telomerase from the telomere.

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Figures

Figure 1.
Figure 1.
Ccq1 C-terminus negatively controls telomere lengthening. (A) Schematic diagram of Ccq1 domains. The protein interaction regions were primarily assessed by the yeast two hybrid system. The core domain of Ccq1 stems from the region 131–441 residues that interacts with Tpz1 and is essential for any function of Ccq1. The Est1 binding domain contains residues including Threonine 93 and the Tpz1-binding domain and therefore begins from 64th amino acids and includes the Tpz1 binding site. The Ccq1 dimerization and Clr3 binding domain is located on the C-terminus (amino acids 500–735), which includes the coiled-coil motifs. (B). Telomere Southern blot shows that truncation of the coiled-coil motifs in Ccq1 leads to slight elongation of telomeres. The ccq1(1–500) and (1–441) truncation mutants exhibited slightly longer telomeres, but further C-terminal truncation or truncation of the Est1 binding domain resulted in maintenance of short telomeres, equivalent to ccq1Δ. EcoRI digested telomere fragments are an average length of 1 kb in wild type cells. (C) Co-immunoprecipitation of the PK epitope-tagged Ccq1 truncations revealed association with Tpz1 and Trt1. Although the Ccq1(131–441) truncation interacted with Tpz1 in the yeast two-hybrid system (Supplementary Figure S2A), neither this interaction nor telomerase association could be detected by co-immunoprecipitation. Slower migrating bands of Tpz1 are presumably caused by phospho-modifications, which are lost in ccq1(1–441) and ccq1(131–441) backgrounds.
Figure 2.
Figure 2.
The Ccq1-Est1 fusion bypasses the recruitment step. (A) Schematic diagram of the fusion system. The ccq1 gene sequence with thirteen tandem myc sequences was inserted after the start codon of one est1 allele in a diploid strain heterozygous for ccq1Δ. Expression of the intact chimera gene product (Ccq1–13xMyc-Est1) through the endogenous est1 promoter was confirmed using anti-Myc antibody (Supplementary Figure S3). After sporulation, haploids were obtained which expressed the chimera protein from the est1 locus in a ccq1Δ or ccq1+ background. (B) Telomere Southern blot shows that expression of the Ccq1-Est1 fusion protein leads to longer telomere maintenance in the absence of endogenous Ccq1. 13xMyc epitope tagging to N-terminus of Est1 and the C-terminus of Ccq1 did not impair function in telomere length homeostasis. (C) Telomere Southern blot shows that the Ccq1-Est1 chimera bypasses need for phosphorylation at Ccq1 Thr93 for telomere maintenance but truncation of Ccq1 N-terminus (131–735) leads to maintenance of short telomeres. In the presence of the Ccq1(131–735)-Est1 chimera, telomere lengthening was severely impaired in ccq1+ background. Nevertheless, elongation of telomeres was observed in the ccq1Δ background, suggesting that negative regulation by endogenous Ccq1 remains functional. (D) Co-immunoprecipitation with the Ccq1–13xMyc-Est1 chimera shows stable association with Trt1. (E) Chromatin immunoprecipitaion (ChIP) with the PK epitope-tagged Trt1 shows efficient recruitment of telomerase to telomeres in the presence of the Ccq1-Est1 chimera and the absence of endogenous Ccq1. DNA fragments containing telomere-adjacent sequence and the act1 gene (as internal control) were detected by quantitative PCR. ChIP efficiencies of telomeric DNA and act1 were calculated against input (whole cell extract), and the fold enrichment over no-tagged negative control is expressed. Mean average of three biological replicas is shown. Significant differences over Trt1-PK (wild type) are indicated as asterisks (two-tailed t-test: *P < 0.05).
Figure 3.
Figure 3.
Telomerase free Ccq1 negatively regulates telomere length via its Tpz1-binding and C-terminal domains. (A) Telomere Southern blot shows that Ccq1 C-terminus truncation (1–441) leads to telomere elongation, like ccq1Δ, in the presence of the Ccq1-Est1 chimera. Conversely, the N-terminus truncation (131–735) leads to telomere shortening but further truncation over the Tpz1 binding domain (139–735 and 500–735) leads to loss of negative regulation. (B) Both the Ccq1-Est1 chimera and the truncated Ccq1 (ΔC: amino acids 1–441; ΔN: amino acids 131–735; Δ: null) were co-immunoprecipitated with the HA epitope-tagged Tpz1. Stable association between the Ccq1-Est1 chimera and Tpz1 was observed in the absence of endogenous Ccq1 (Δ) and in the presence of the C-terminus truncated Ccq1 (ΔC). Slower migrating bands of Tpz1 are presumably caused by phospho-modifications, which are lost in ccq1Δ and ccq1(1–441) backgrounds that cause a telomere elongation phenotype. (C) Schematic diagram describing Ccq1 association with Tpz1 in cells expressing both endogenous (full-length or truncated) Ccq1, and the Ccq1-Est1 chimera. The telomere status is indicated in the brackets. The presence of free Ccq1 weakens the association between Tpz1 and the Ccq1-Est1 chimera (represented as dashed lines). This inhibitory effect is reduced by Ccq1 with a C-terminal deletion (represented as thinner block dash line).
Figure 4.
Figure 4.
The coiled-coil motifs region of Ccq1 forms a homodimer/multimer. (A) The yeast two-hybrid assay shows that the C-terminal coiled-coil motifs (500–735) forms a dimer/multimer. The indicated Ccq1 truncation proteins were fused to the GAL4 activation domain (AD) and the C-terminus fragments of Ccq1 were fused to the GAL4 DNA binding domain (BD). Selection plate lacks adenine and histidine. (B) The Ccq1-Est1 chimera and Tpz1 were co-immunoprecipitated with the FLAG epitope-tagged truncated Ccq1 (ΔC: 1–441aa; ΔN: 131–735aa). While Tpz1 interacted with both Ccq1 truncations, the C-terminus truncation failed to interact with the Ccq1-Est1 chimera. (C) Summary representation of co-immunoprecipitation study in B and D. Ccq1-Flag and Ccq1-Myc-Est1 interacts via their Ccq1 C-terminal domain. (D) Co-immunoprecipitation of the Myc epitope-tagged Est1 and the Ccq1-Est1 chimera (ΔC: Est1 is fused to Ccq1(1–441), the C-terminus truncation) showed that the Ccq1-Est1 chimera associated with Ccq1 via its C-terminus. (E) Telomere Southern blot shows that truncation of the Ccq1 C-terminus region within the Ccq1-Est1 chimera did not impair telomere lengthening. The elongation of telomeres by the Ccq1(1–441)-fused-Est1 chimera was similar to that by full length Ccq1-Est1 chimera in the absence of endogenous Ccq1.
Figure 5.
Figure 5.
Ccq1 dependent negative regulation of telomere lengthening via Clr3-SHREC. (A) The yeast two-hybrid assay shows that Clr3 interacts strongly with Ccq1(500–735) and weakly with Ccq1(1–441). The indicated Ccq1 truncation proteins were fused to the GAL4 activation domain (AD) and Tpz1 and Clr3 were fused to the GAL4 DNA binding domain (BD). Selection plate lacks adenine and histidine. (B) Co-immunoprecipitation with the truncated Ccq1 showed that endogenously expressed Ccq1 associates with Clr3 via its C-terminus (amino acids 500–735). (C) Telomere Southern blot shows mild telomere elongation in clr4Δ, similar to ccq1(1–441). Deletion of clr3 and mit1 leads to elongation of telomeres only in the presence of the Ccq1-Est1 chimera.
Figure 6.
Figure 6.
Clr3 associates with Est1-unbound Ccq1 and represses telomere-adjacent transcription. (A) Clr3 was co-immunoprecipitated with the Myc-epitope tagged Ccq1 but not with the Ccq1-Est1 chimera (in ccq1Δ background). Presence of endogenous Est1-free Ccq1 further reduced the interaction between Clr3 and the Ccq1-Est1 chimera (Supplementary Figure S10). (B) The heterochromatic gene-silencing assay. Serial dilution spot assay to measure transcription efficiencies of genes inserted at the centromere and telomere. The wild type ade6+ gene cassette was inserted at a centromeric outer repeat region of chromosome 1, and the wild type ura4+ gene cassette was inserted at the telomere on the left arm of chromosome 2. Centromeric repression of the ade6+ gene leads to a growth defect and red colouring, and telomeric repression of the ura4+ gene leads to survival of cells under 5-FOA treatment. Control cells are prototrophic, expressing both Ade6 and Ura4 (lane 1). The taz1 deletion or ccq1(1–441) truncation mutation leads to activation of telomeric transcription, whereas deletion of clr3 or clr4 leads to gene silencing defects at both telomeres and centromeres.
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