Limits...
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.

Show MeSH
Eic1 and Eic2 depend on distinct Cnp1CENP-A assembly factors for their association with centromeres. (a,b) Eic1 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2 and Scm3HJURP, but is largely independent of Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic2. qChIP analyses of Eic1-GFP association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a), or when grown at 32°C (b). (c,d) Eic2 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2, Scm3HJURP, Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic1. qChIP analyses of Eic2-GFP 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 in (a,c,d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (b). Error bars represent standard deviation between at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043117&req=5

RSOB140043F6: Eic1 and Eic2 depend on distinct Cnp1CENP-A assembly factors for their association with centromeres. (a,b) Eic1 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2 and Scm3HJURP, but is largely independent of Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic2. qChIP analyses of Eic1-GFP association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a), or when grown at 32°C (b). (c,d) Eic2 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2, Scm3HJURP, Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic1. qChIP analyses of Eic2-GFP 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 in (a,c,d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (b). Error bars represent standard deviation between at least three biological replicates.

Mentions: Previous analyses in fission yeast have shown that Mis16RbAp46/48/Hat2 and Mis18 are required for the localization of the Cnp1CENP-A-specific chaperone Scm3HJURP and the CCAN protein Mis6CENP-I/Ctf3 to centromeres [9,11,12]. Moreover, the localization of Mis18 at centromeres is unaffected by mutations in Cnp1CENP-A, Mis6CENP-I/Ctf3 or Scm3HJURP, whereas Scm3HJURP is dependent on functional Mis6CENP-I/Ctf3, Mis16RbAp46/48/Hat2 and Mis18 for its centromeric localization [9,11,12]. Such analyses suggest that Mis18, and probably Mis16RbAp46/48/Hat2, function to mediate the recruitment of the Cnp1CENP-A assembly factors Mis6CENP-I/Ctf3 and Scm3HJURP to centromeres and thus the incorporation of Cnp1CENP-A itself. To further dissect the role of Eic1 and Eic2, and to determine whether they act together with Mis16RbAp46/48/Hat2 and Mis18, we used qChIP to examine the dependencies of Eic1 and Eic2 for centromere localization in strains expressing various ts kinetochore proteins. Eic1 association with centromeres was found to be entirely dependent on functional Mis18, as indicated by undetectable levels of Eic1 at centromeres in mis18-262 cells grown at 36°C (figure 6a and electronic supplementary material, figure S7a). Lower Eic1 levels were also detected on centromeric central cores in mis16-53 and scm3-139 mutants at restrictive temperature; however, they were largely unchanged in cells expressing mutant Cnp1CENP-A, Mis6CENP-I/Ctf3 and Mis12 proteins (figure 6a and electronic supplementary material, figure S7a). Centromeric Eic1 levels also remained unaffected in eic2Δ cells (figure 6b). Thus, the localization of Eic1 at kinetochores is mainly dependent on its partner proteins Mis16RbAp46/48/Hat2 and Mis18, but not Eic2.Figure 6.


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 and Eic2 depend on distinct Cnp1CENP-A assembly factors for their association with centromeres. (a,b) Eic1 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2 and Scm3HJURP, but is largely independent of Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic2. qChIP analyses of Eic1-GFP association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a), or when grown at 32°C (b). (c,d) Eic2 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2, Scm3HJURP, Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic1. qChIP analyses of Eic2-GFP 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 in (a,c,d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (b). Error bars represent standard deviation between at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140043F6: Eic1 and Eic2 depend on distinct Cnp1CENP-A assembly factors for their association with centromeres. (a,b) Eic1 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2 and Scm3HJURP, but is largely independent of Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic2. qChIP analyses of Eic1-GFP association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a), or when grown at 32°C (b). (c,d) Eic2 association with centromeres is dependent on Mis18, Mis16RbAp46/48/Hat2, Scm3HJURP, Cnp1CENP-A, Mis6CENP-I/Ctf3, Mis12 and Eic1. qChIP analyses of Eic2-GFP 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 in (a,c,d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (b). Error bars represent standard deviation between at least three biological replicates.
Mentions: Previous analyses in fission yeast have shown that Mis16RbAp46/48/Hat2 and Mis18 are required for the localization of the Cnp1CENP-A-specific chaperone Scm3HJURP and the CCAN protein Mis6CENP-I/Ctf3 to centromeres [9,11,12]. Moreover, the localization of Mis18 at centromeres is unaffected by mutations in Cnp1CENP-A, Mis6CENP-I/Ctf3 or Scm3HJURP, whereas Scm3HJURP is dependent on functional Mis6CENP-I/Ctf3, Mis16RbAp46/48/Hat2 and Mis18 for its centromeric localization [9,11,12]. Such analyses suggest that Mis18, and probably Mis16RbAp46/48/Hat2, function to mediate the recruitment of the Cnp1CENP-A assembly factors Mis6CENP-I/Ctf3 and Scm3HJURP to centromeres and thus the incorporation of Cnp1CENP-A itself. To further dissect the role of Eic1 and Eic2, and to determine whether they act together with Mis16RbAp46/48/Hat2 and Mis18, we used qChIP to examine the dependencies of Eic1 and Eic2 for centromere localization in strains expressing various ts kinetochore proteins. Eic1 association with centromeres was found to be entirely dependent on functional Mis18, as indicated by undetectable levels of Eic1 at centromeres in mis18-262 cells grown at 36°C (figure 6a and electronic supplementary material, figure S7a). Lower Eic1 levels were also detected on centromeric central cores in mis16-53 and scm3-139 mutants at restrictive temperature; however, they were largely unchanged in cells expressing mutant Cnp1CENP-A, Mis6CENP-I/Ctf3 and Mis12 proteins (figure 6a and electronic supplementary material, figure S7a). Centromeric Eic1 levels also remained unaffected in eic2Δ cells (figure 6b). Thus, the localization of Eic1 at kinetochores is mainly dependent on its partner proteins Mis16RbAp46/48/Hat2 and Mis18, but not Eic2.Figure 6.

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