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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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The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 influence Eic1 association with centromeres. (a) Fta7-Myc, (b) Cnl2-Myc and (c) Mal2-GFP association with centromeres is partly dependent on Eic1. (d) Eic1-GFP association with centromeres is greatly dependent on Mal2. qChIP analyses of (a) Fta7-Myc, (b) Cnl2-Myc, (c) Mal2-GFP and (d) Eic1-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. Error bars represent standard deviation between at least three biological replicates. (e) eic1-1 displays a severe negative genetic interaction when combined with mal2-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (f) A model for Cnp1CENP-A maintenance at S. pombe centromeres mediated by Eic1. The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 that are constitutively bound to centromeres together form a module that recruits Eic1, and thereby regulates the temporal association of Eic1, Mis16RbAp46/48/Hat2, Mis18 and Eic2 with centromeres. Once bound, Mis16RbAp46/48/Hat2/Mis18 then likely recruit the Cnp1CENP-A-specific chaperone Scm3HJURP to centromeres (the dashed arrow indicates that only an in vitro association between these proteins has been demonstrated), and thus ensures replenishment of Cnp1CENP-A (‘A’ in closed oval) at centromeres in every cell cycle.
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RSOB140043F8: The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 influence Eic1 association with centromeres. (a) Fta7-Myc, (b) Cnl2-Myc and (c) Mal2-GFP association with centromeres is partly dependent on Eic1. (d) Eic1-GFP association with centromeres is greatly dependent on Mal2. qChIP analyses of (a) Fta7-Myc, (b) Cnl2-Myc, (c) Mal2-GFP and (d) Eic1-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. Error bars represent standard deviation between at least three biological replicates. (e) eic1-1 displays a severe negative genetic interaction when combined with mal2-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (f) A model for Cnp1CENP-A maintenance at S. pombe centromeres mediated by Eic1. The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 that are constitutively bound to centromeres together form a module that recruits Eic1, and thereby regulates the temporal association of Eic1, Mis16RbAp46/48/Hat2, Mis18 and Eic2 with centromeres. Once bound, Mis16RbAp46/48/Hat2/Mis18 then likely recruit the Cnp1CENP-A-specific chaperone Scm3HJURP to centromeres (the dashed arrow indicates that only an in vitro association between these proteins has been demonstrated), and thus ensures replenishment of Cnp1CENP-A (‘A’ in closed oval) at centromeres in every cell cycle.

Mentions: To further investigate the functional niche of Eic1 and Eic2, immunoprecipitates of both GFP-tagged proteins were subjected to LC-MS/MS analyses to identify associated factors. Only Eic1 and Mis18 were detected in Eic2-GFP immunoprecipitates (figure 7a). However, three CCAN/Mis6/Ctf19 complex components were found to associate with Eic1-GFP: Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 (figure 7a). Co-immunoprecipitation of Myc-tagged Fta7CENP-Q/Okp1 or Cnl2Nkp2 with Eic1-GFP verified that these CCAN/Mis6/Ctf19 complex components associate with Eic1 (figure 7b). ChIP analyses revealed that the association of Myc-tagged Fta7CENP-Q/Okp1, Myc-tagged Cnl2Nkp2 and GFP-tagged Mal2CENP-O/Mcm21 with centromeres is only partially dependent on functional Eic1 (figure 8a–c), as these proteins are detectable to a significant degree at centromeres in eic1-1 cells even at non-permissive temperature. This is consistent with the finding that centromeric association of the CCAN component Mis6CENP-I/Ctf3 also only partly depends on Eic1 function (figure 4d).Figure 7.


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)

The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 influence Eic1 association with centromeres. (a) Fta7-Myc, (b) Cnl2-Myc and (c) Mal2-GFP association with centromeres is partly dependent on Eic1. (d) Eic1-GFP association with centromeres is greatly dependent on Mal2. qChIP analyses of (a) Fta7-Myc, (b) Cnl2-Myc, (c) Mal2-GFP and (d) Eic1-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. Error bars represent standard deviation between at least three biological replicates. (e) eic1-1 displays a severe negative genetic interaction when combined with mal2-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (f) A model for Cnp1CENP-A maintenance at S. pombe centromeres mediated by Eic1. The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 that are constitutively bound to centromeres together form a module that recruits Eic1, and thereby regulates the temporal association of Eic1, Mis16RbAp46/48/Hat2, Mis18 and Eic2 with centromeres. Once bound, Mis16RbAp46/48/Hat2/Mis18 then likely recruit the Cnp1CENP-A-specific chaperone Scm3HJURP to centromeres (the dashed arrow indicates that only an in vitro association between these proteins has been demonstrated), and thus ensures replenishment of Cnp1CENP-A (‘A’ in closed oval) at centromeres in every cell cycle.
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Related In: Results  -  Collection

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RSOB140043F8: The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 influence Eic1 association with centromeres. (a) Fta7-Myc, (b) Cnl2-Myc and (c) Mal2-GFP association with centromeres is partly dependent on Eic1. (d) Eic1-GFP association with centromeres is greatly dependent on Mal2. qChIP analyses of (a) Fta7-Myc, (b) Cnl2-Myc, (c) Mal2-GFP and (d) Eic1-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. Error bars represent standard deviation between at least three biological replicates. (e) eic1-1 displays a severe negative genetic interaction when combined with mal2-1. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (f) A model for Cnp1CENP-A maintenance at S. pombe centromeres mediated by Eic1. The CCAN components Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 that are constitutively bound to centromeres together form a module that recruits Eic1, and thereby regulates the temporal association of Eic1, Mis16RbAp46/48/Hat2, Mis18 and Eic2 with centromeres. Once bound, Mis16RbAp46/48/Hat2/Mis18 then likely recruit the Cnp1CENP-A-specific chaperone Scm3HJURP to centromeres (the dashed arrow indicates that only an in vitro association between these proteins has been demonstrated), and thus ensures replenishment of Cnp1CENP-A (‘A’ in closed oval) at centromeres in every cell cycle.
Mentions: To further investigate the functional niche of Eic1 and Eic2, immunoprecipitates of both GFP-tagged proteins were subjected to LC-MS/MS analyses to identify associated factors. Only Eic1 and Mis18 were detected in Eic2-GFP immunoprecipitates (figure 7a). However, three CCAN/Mis6/Ctf19 complex components were found to associate with Eic1-GFP: Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 (figure 7a). Co-immunoprecipitation of Myc-tagged Fta7CENP-Q/Okp1 or Cnl2Nkp2 with Eic1-GFP verified that these CCAN/Mis6/Ctf19 complex components associate with Eic1 (figure 7b). ChIP analyses revealed that the association of Myc-tagged Fta7CENP-Q/Okp1, Myc-tagged Cnl2Nkp2 and GFP-tagged Mal2CENP-O/Mcm21 with centromeres is only partially dependent on functional Eic1 (figure 8a–c), as these proteins are detectable to a significant degree at centromeres in eic1-1 cells even at non-permissive temperature. This is consistent with the finding that centromeric association of the CCAN component Mis6CENP-I/Ctf3 also only partly depends on Eic1 function (figure 4d).Figure 7.

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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