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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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Analysis of genetic interactions between eic1 or eic2 mutants and mutations in Cnp1CENP-A or Cnp1CENP-A assembly factors. (a) eic1+:hygR and eic1-1 cells display reduced growth when combined with mutations in mis18, scm3, cnp1 or mis6. (b) eic2Δ cells display genetic interactions when combined with mis18-262 and scm3-139, but not eic1-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (c) A tabular summary of genetic interactions analysed in this study. s.s, synthetic sick/reduced growth; s.l, synthetic lethal; n.d., not determined; —, no interaction detected.
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RSOB140043F5: Analysis of genetic interactions between eic1 or eic2 mutants and mutations in Cnp1CENP-A or Cnp1CENP-A assembly factors. (a) eic1+:hygR and eic1-1 cells display reduced growth when combined with mutations in mis18, scm3, cnp1 or mis6. (b) eic2Δ cells display genetic interactions when combined with mis18-262 and scm3-139, but not eic1-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (c) A tabular summary of genetic interactions analysed in this study. s.s, synthetic sick/reduced growth; s.l, synthetic lethal; n.d., not determined; —, no interaction detected.

Mentions: Epistasis analyses (double mutant combinations) frequently reveal positive and negative genetic interactions between mutants and consequently inform on the functional niche of specific proteins. The eic1-1 mutation exhibited significantly reduced growth when combined with ts mutations in mis18, scm3, cnp1 and mis6 (figure 5a and electronic supplementary material, figure S6a; summarized in figure 5c). We failed to generate eic1-1 mis16-53 and eic1-1 scm3-139 double mutant strains, suggesting that eic1-1 has a synthetic lethal interaction with both mis16-53 and scm3-139. Surprisingly, such synthetic interactions could also be observed for the eic1+:hygR allele, which must compromise eic1 function in sensitized genetic backgrounds and therefore be considered a hypomorphic allele of eic1 (figure 5 and electronic supplementary material, figure S6a). These results, in conjunction with our ChIP analyses of Cnp1CENP-A assembly factors (figure 4a–d), clearly demonstrate that Eic1 makes important contributions to the proper localization of Cnp1CENP-A assembly factors and Cnp1CENP-A itself.Figure 5.


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)

Analysis of genetic interactions between eic1 or eic2 mutants and mutations in Cnp1CENP-A or Cnp1CENP-A assembly factors. (a) eic1+:hygR and eic1-1 cells display reduced growth when combined with mutations in mis18, scm3, cnp1 or mis6. (b) eic2Δ cells display genetic interactions when combined with mis18-262 and scm3-139, but not eic1-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (c) A tabular summary of genetic interactions analysed in this study. s.s, synthetic sick/reduced growth; s.l, synthetic lethal; n.d., not determined; —, no interaction detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043117&req=5

RSOB140043F5: Analysis of genetic interactions between eic1 or eic2 mutants and mutations in Cnp1CENP-A or Cnp1CENP-A assembly factors. (a) eic1+:hygR and eic1-1 cells display reduced growth when combined with mutations in mis18, scm3, cnp1 or mis6. (b) eic2Δ cells display genetic interactions when combined with mis18-262 and scm3-139, but not eic1-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (c) A tabular summary of genetic interactions analysed in this study. s.s, synthetic sick/reduced growth; s.l, synthetic lethal; n.d., not determined; —, no interaction detected.
Mentions: Epistasis analyses (double mutant combinations) frequently reveal positive and negative genetic interactions between mutants and consequently inform on the functional niche of specific proteins. The eic1-1 mutation exhibited significantly reduced growth when combined with ts mutations in mis18, scm3, cnp1 and mis6 (figure 5a and electronic supplementary material, figure S6a; summarized in figure 5c). We failed to generate eic1-1 mis16-53 and eic1-1 scm3-139 double mutant strains, suggesting that eic1-1 has a synthetic lethal interaction with both mis16-53 and scm3-139. Surprisingly, such synthetic interactions could also be observed for the eic1+:hygR allele, which must compromise eic1 function in sensitized genetic backgrounds and therefore be considered a hypomorphic allele of eic1 (figure 5 and electronic supplementary material, figure S6a). These results, in conjunction with our ChIP analyses of Cnp1CENP-A assembly factors (figure 4a–d), clearly demonstrate that Eic1 makes important contributions to the proper localization of Cnp1CENP-A assembly factors and Cnp1CENP-A itself.Figure 5.

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.

Show MeSH
Related in: MedlinePlus