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Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

Subramanian L, Toda NR, Rappsilber J, Allshire RC - Open Biol (2014)

Bottom Line: CENP-A chromatin forms the foundation for kinetochore assembly.Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors.The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

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Eic1 is required for Cnp1CENP-A assembly, while Eic2 is dispensable. (a) ts mutations in eic1 affect cell viability, while eic2Δ cells show no defects in growth. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (b) eic1 mutants display reduced Cnp1CENP-A levels at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h. Enrichment of cc2 DNA relative to the act1 locus is presented. (c) eic1 mutants display sensitivity to TBZ, while eic2Δ cells show no TBZ sensitivity. Five-fold serial dilutions of cells spotted on YES media (untreated) or YES media supplemented with 12.5 μg ml−1 TBZ, and incubated at 25°C. (d) eic2Δ cells display no loss of Cnp1CENP-A at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at 32°C. Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented. Error bars in (b,d) represent standard deviation between at least three biological replicates.
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RSOB140043F3: Eic1 is required for Cnp1CENP-A assembly, while Eic2 is dispensable. (a) ts mutations in eic1 affect cell viability, while eic2Δ cells show no defects in growth. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (b) eic1 mutants display reduced Cnp1CENP-A levels at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h. Enrichment of cc2 DNA relative to the act1 locus is presented. (c) eic1 mutants display sensitivity to TBZ, while eic2Δ cells show no TBZ sensitivity. Five-fold serial dilutions of cells spotted on YES media (untreated) or YES media supplemented with 12.5 μg ml−1 TBZ, and incubated at 25°C. (d) eic2Δ cells display no loss of Cnp1CENP-A at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at 32°C. Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented. Error bars in (b,d) represent standard deviation between at least three biological replicates.

Mentions: Mis18 and associated proteins have been shown to be required to maintain CENP-A at centromeres in fission yeast and vertebrates [11,13,37]. If Eic1 and Eic2 are critical for Mis18 function then they should also be required to maintain normal Cnp1CENP-A levels at centromeres. As the eic1+ gene is essential for cell viability, we generated a conditional temperature-sensitive (ts) mutant of eic1 (eic1-1:hygR, hereafter referred to as eic1-1) in which a single amino acid substitution (Phe102Ser) at a conserved residue rendered cells inviable at 36°C but with retained viability at 25°C (figure 3a). We also generated a corresponding wild-type allele of eic1 (eic1+:hygR) as a control in which, as with eic1-1, a hygromycin resistance marker was inserted within the 3′UTR of eic1 at its endogenous locus for ease of genetic manipulation (figure 3a). qChIP analyses demonstrated that Cnp1CENP-A levels were significantly diminished at centromeres in eic1-1 cells at restrictive temperature (figure 3b; electronic supplementary material, figure S3a). Cells harbouring a second ts allele of eic1 (eic1-2:hygR; two substituted residues, Lys37Glu and Tyr55His, hereafter referred to as eic1-2) also demonstrated significant loss of Cnp1CENP-A from centromeres at 36°C (figure 3a,b; electronic supplementary material, figure S3a). Furthermore, eic1-1 and eic1-2 cells displayed sensitivity to the microtubule depolymerizing drug thiabendazole (TBZ), whereas eic1+:hygR and the mis18-262 ts mutant did not confer TBZ sensitivity (figure 3c). TBZ sensitivity suggests that kinetochore–microtubule interactions are defective in eic1 mutants. Aberrant kinetochore function in eic1-1 cells was supported by cytological analyses, which revealed severe chromosome segregation defects (electronic supplementary material, figure S3b). qChIP analyses demonstrated that GFP-tagged Eic1-1 and Eic1-2 mutant proteins remained associated with centromeres even at 36°C; thus, the observed phenotypes of eic1-1 and eic1-2 cells are not a consequence of the complete absence of Eic1 protein at centromeres (electronic supplementary material, figure S3c).Figure 3.


Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

Subramanian L, Toda NR, Rappsilber J, Allshire RC - Open Biol (2014)

Eic1 is required for Cnp1CENP-A assembly, while Eic2 is dispensable. (a) ts mutations in eic1 affect cell viability, while eic2Δ cells show no defects in growth. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (b) eic1 mutants display reduced Cnp1CENP-A levels at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h. Enrichment of cc2 DNA relative to the act1 locus is presented. (c) eic1 mutants display sensitivity to TBZ, while eic2Δ cells show no TBZ sensitivity. Five-fold serial dilutions of cells spotted on YES media (untreated) or YES media supplemented with 12.5 μg ml−1 TBZ, and incubated at 25°C. (d) eic2Δ cells display no loss of Cnp1CENP-A at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at 32°C. Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented. Error bars in (b,d) represent standard deviation between at least three biological replicates.
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Related In: Results  -  Collection

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RSOB140043F3: Eic1 is required for Cnp1CENP-A assembly, while Eic2 is dispensable. (a) ts mutations in eic1 affect cell viability, while eic2Δ cells show no defects in growth. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (b) eic1 mutants display reduced Cnp1CENP-A levels at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h. Enrichment of cc2 DNA relative to the act1 locus is presented. (c) eic1 mutants display sensitivity to TBZ, while eic2Δ cells show no TBZ sensitivity. Five-fold serial dilutions of cells spotted on YES media (untreated) or YES media supplemented with 12.5 μg ml−1 TBZ, and incubated at 25°C. (d) eic2Δ cells display no loss of Cnp1CENP-A at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at 32°C. Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented. Error bars in (b,d) represent standard deviation between at least three biological replicates.
Mentions: Mis18 and associated proteins have been shown to be required to maintain CENP-A at centromeres in fission yeast and vertebrates [11,13,37]. If Eic1 and Eic2 are critical for Mis18 function then they should also be required to maintain normal Cnp1CENP-A levels at centromeres. As the eic1+ gene is essential for cell viability, we generated a conditional temperature-sensitive (ts) mutant of eic1 (eic1-1:hygR, hereafter referred to as eic1-1) in which a single amino acid substitution (Phe102Ser) at a conserved residue rendered cells inviable at 36°C but with retained viability at 25°C (figure 3a). We also generated a corresponding wild-type allele of eic1 (eic1+:hygR) as a control in which, as with eic1-1, a hygromycin resistance marker was inserted within the 3′UTR of eic1 at its endogenous locus for ease of genetic manipulation (figure 3a). qChIP analyses demonstrated that Cnp1CENP-A levels were significantly diminished at centromeres in eic1-1 cells at restrictive temperature (figure 3b; electronic supplementary material, figure S3a). Cells harbouring a second ts allele of eic1 (eic1-2:hygR; two substituted residues, Lys37Glu and Tyr55His, hereafter referred to as eic1-2) also demonstrated significant loss of Cnp1CENP-A from centromeres at 36°C (figure 3a,b; electronic supplementary material, figure S3a). Furthermore, eic1-1 and eic1-2 cells displayed sensitivity to the microtubule depolymerizing drug thiabendazole (TBZ), whereas eic1+:hygR and the mis18-262 ts mutant did not confer TBZ sensitivity (figure 3c). TBZ sensitivity suggests that kinetochore–microtubule interactions are defective in eic1 mutants. Aberrant kinetochore function in eic1-1 cells was supported by cytological analyses, which revealed severe chromosome segregation defects (electronic supplementary material, figure S3b). qChIP analyses demonstrated that GFP-tagged Eic1-1 and Eic1-2 mutant proteins remained associated with centromeres even at 36°C; thus, the observed phenotypes of eic1-1 and eic1-2 cells are not a consequence of the complete absence of Eic1 protein at centromeres (electronic supplementary material, figure S3c).Figure 3.

Bottom Line: CENP-A chromatin forms the foundation for kinetochore assembly.Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors.The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

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