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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 and Eic2 promote normal levels of association of Cnp1CENP-A assembly factors with centromeres. (a,b) Mis18-GFP and Mis16-GFP association with centromeres is entirely dependent on Eic1, and (e,f) partly dependent on Eic2. (c) Scm3-GFP association with centromeres is dependent on Eic1, and (g) largely independent of Eic2. (d) Mis6-HA association with centromeres is partly dependent on Eic1, and (h) Eic2. qChIP analyses of (a,e) Mis18-GFP, (b,f) Mis16-GFP, (c,g) Scm3-GFP and (d,h) Mis6-HA association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a–d); or at 32°C (e–h). Enrichment of cc2 DNA relative to the act1 locus is presented in (a–d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (e–h). Error bars represent standard deviation between at least three biological replicates.
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RSOB140043F4: Eic1 and Eic2 promote normal levels of association of Cnp1CENP-A assembly factors with centromeres. (a,b) Mis18-GFP and Mis16-GFP association with centromeres is entirely dependent on Eic1, and (e,f) partly dependent on Eic2. (c) Scm3-GFP association with centromeres is dependent on Eic1, and (g) largely independent of Eic2. (d) Mis6-HA association with centromeres is partly dependent on Eic1, and (h) Eic2. qChIP analyses of (a,e) Mis18-GFP, (b,f) Mis16-GFP, (c,g) Scm3-GFP and (d,h) Mis6-HA association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a–d); or at 32°C (e–h). Enrichment of cc2 DNA relative to the act1 locus is presented in (a–d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (e–h). Error bars represent standard deviation between at least three biological replicates.

Mentions: Previous studies have shown that defective Mis16RbAp46/48/Hat2 or Mis18 function affects the localization of the Cnp1CENP-A loading factors Scm3HJURP and Mis6CENP-I/Ctf3 at centromeres [9,11,12]. Vertebrate Mis18α/β and Mis18BP1KNL2 have also been shown to be required for the recruitment of the CENP-A chaperone HJURPScm3 to centromeres, and consequently the incorporation of CENP-A at centromeres [13,25]. We find that the levels of GFP-tagged Mis16RbAp46/48/Hat2 and Mis18 associated with fission yeast centromeres are greatly reduced in the eic1-1 mutant even at 25°C (figure 4a,b), consistent with the observed synthetic genetic interaction between eic1-1 and the GFP-tagged alleles of mis16 and mis18 (electronic supplementary material, figure S5). Additionally, we find that Scm3HJURP association with centromeres is also dependent on Eic1 function (figure 4c). Interestingly, we find Mis6CENP-I/Ctf3 association with centromeres to be only partially dependent on Eic1 (figure 4d), as intermediate levels of Mis6CENP-I/Ctf3 are retained at centromeres even when Eic1 function is compromised. Thus Eic1, in conjunction with its interacting partners Mis16RbAp46/48/Hat2 and Mis18, is essential to maintain normal levels of Cnp1CENP-A as well as Cnp1CENP-A loading factors on centromeres.Figure 4.


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 promote normal levels of association of Cnp1CENP-A assembly factors with centromeres. (a,b) Mis18-GFP and Mis16-GFP association with centromeres is entirely dependent on Eic1, and (e,f) partly dependent on Eic2. (c) Scm3-GFP association with centromeres is dependent on Eic1, and (g) largely independent of Eic2. (d) Mis6-HA association with centromeres is partly dependent on Eic1, and (h) Eic2. qChIP analyses of (a,e) Mis18-GFP, (b,f) Mis16-GFP, (c,g) Scm3-GFP and (d,h) Mis6-HA association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a–d); or at 32°C (e–h). Enrichment of cc2 DNA relative to the act1 locus is presented in (a–d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (e–h). Error bars represent standard deviation between at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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RSOB140043F4: Eic1 and Eic2 promote normal levels of association of Cnp1CENP-A assembly factors with centromeres. (a,b) Mis18-GFP and Mis16-GFP association with centromeres is entirely dependent on Eic1, and (e,f) partly dependent on Eic2. (c) Scm3-GFP association with centromeres is dependent on Eic1, and (g) largely independent of Eic2. (d) Mis6-HA association with centromeres is partly dependent on Eic1, and (h) Eic2. qChIP analyses of (a,e) Mis18-GFP, (b,f) Mis16-GFP, (c,g) Scm3-GFP and (d,h) Mis6-HA association with centromeres in the indicated strains when grown at permissive (25°C) versus restrictive temperature (36°C) for 8 h (a–d); or at 32°C (e–h). Enrichment of cc2 DNA relative to the act1 locus is presented in (a–d). Enrichment of cc2 or cc1/3 DNA relative to the act1 locus is presented in (e–h). Error bars represent standard deviation between at least three biological replicates.
Mentions: Previous studies have shown that defective Mis16RbAp46/48/Hat2 or Mis18 function affects the localization of the Cnp1CENP-A loading factors Scm3HJURP and Mis6CENP-I/Ctf3 at centromeres [9,11,12]. Vertebrate Mis18α/β and Mis18BP1KNL2 have also been shown to be required for the recruitment of the CENP-A chaperone HJURPScm3 to centromeres, and consequently the incorporation of CENP-A at centromeres [13,25]. We find that the levels of GFP-tagged Mis16RbAp46/48/Hat2 and Mis18 associated with fission yeast centromeres are greatly reduced in the eic1-1 mutant even at 25°C (figure 4a,b), consistent with the observed synthetic genetic interaction between eic1-1 and the GFP-tagged alleles of mis16 and mis18 (electronic supplementary material, figure S5). Additionally, we find that Scm3HJURP association with centromeres is also dependent on Eic1 function (figure 4c). Interestingly, we find Mis6CENP-I/Ctf3 association with centromeres to be only partially dependent on Eic1 (figure 4d), as intermediate levels of Mis6CENP-I/Ctf3 are retained at centromeres even when Eic1 function is compromised. Thus Eic1, in conjunction with its interacting partners Mis16RbAp46/48/Hat2 and Mis18, is essential to maintain normal levels of Cnp1CENP-A as well as Cnp1CENP-A loading factors on centromeres.Figure 4.

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