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 associate specifically with centromeres. (a) Eic1 and Eic2 bind the central domain of S. pombe centromeres. qChIP analyses showing enrichments of GFP-tagged Eic1 and Eic2 at the central cores (cc) of centromeres 1, 2 and 3 and imr repeats of centromere 1, relative to the act1 locus. Error bars represent standard deviation between at least three biological replicates. (b) Eic1 and Eic2 exhibit very similar genome-wide association profiles as Mis18 and Scm3HJURP. A comparison between the ChIP-seq profiles of GFP-tagged Eic1, Eic2, Mis18 and Scm3 across centromere 2 is presented, alongside a schematic diagram of centromere 2 (bottom). Normalized coverage represents the number of sequencing fragments obtained from anti-GFP IP normalized to that obtained from the input. (c) Eic1 and Eic2 exhibit very similar cell-cycle localization dynamics as Mis18 and Scm3HJURP. Immunofluorescence of S. pombe cells expressing GFP-tagged Eic1 or Eic2 stained with antibodies to GFP (green) and Cnp1CENP-A (red), and DAPI (blue). Both Eic1 and Eic2 dissociate from centromeres during prometaphase to mid-anaphase of mitosis ((iii)–(vii)) and subsequently reassociate. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140043F2: Eic1 and Eic2 associate specifically with centromeres. (a) Eic1 and Eic2 bind the central domain of S. pombe centromeres. qChIP analyses showing enrichments of GFP-tagged Eic1 and Eic2 at the central cores (cc) of centromeres 1, 2 and 3 and imr repeats of centromere 1, relative to the act1 locus. Error bars represent standard deviation between at least three biological replicates. (b) Eic1 and Eic2 exhibit very similar genome-wide association profiles as Mis18 and Scm3HJURP. A comparison between the ChIP-seq profiles of GFP-tagged Eic1, Eic2, Mis18 and Scm3 across centromere 2 is presented, alongside a schematic diagram of centromere 2 (bottom). Normalized coverage represents the number of sequencing fragments obtained from anti-GFP IP normalized to that obtained from the input. (c) Eic1 and Eic2 exhibit very similar cell-cycle localization dynamics as Mis18 and Scm3HJURP. Immunofluorescence of S. pombe cells expressing GFP-tagged Eic1 or Eic2 stained with antibodies to GFP (green) and Cnp1CENP-A (red), and DAPI (blue). Both Eic1 and Eic2 dissociate from centromeres during prometaphase to mid-anaphase of mitosis ((iii)–(vii)) and subsequently reassociate. Scale bar, 5 μm.

Mentions: CENP-A and all known fission yeast kinetochore proteins localize specifically at centromeres and are enriched over the central domain region of centromeres. Both Eic1 and Eic2 associate with Mis18, which is known to localize to fission yeast centromeres for most of the cell cycle, apart from early prophase to mid-anaphase of mitosis [13]. To examine the localization of Eic1 and Eic2, the endogenous genes expressing Eic1 and Eic2 were fused to GFP. Quantitative chromatin immunoprecipitation (qChIP) analyses demonstrated that, as with Mis18 and other kinetochore proteins, both Eic1-GFP and Eic2-GFP are enriched over the central kinetochore domain (central core plus imr repeats) of fission yeast centromeres (figure 2a). Genome-wide ChIP-seq analyses confirmed this finding and also demonstrated that Eic1-GFP and Eic2-GFP, much like Mis18-GFP and Scm3-GFP, are undetectable at other regions of the genome including the heterochromatic outer repeats of centromeres (figure 2b and electronic supplementary material, figure S2). Immunostaining revealed that both Eic1 and Eic2 GFP-tagged proteins co-localize with Cnp1CENP-A at clustered centromeres in interphase cells (figure 2c(i),(ii)). However, as with Mis16RbAp46/48/Hat2 and Mis18 [11,13], Eic1-GFP and Eic2-GFP dissociate from centromeres in early mitosis (prometaphase), just as the centromeres (Cnp1) begin to form two separate clusters as they biorient on the spindle (figure 2c(iii),(iv)). Moreover, both Eic1-GFP and Eic2-GFP reassociate with centromeres in mid-anaphase when centromeres (Cnp1) and chromosomes (DAPI) have clearly segregated to opposite spindle poles (figure 2c(viii),(ix)). Apart from Mis16RbAp46/48/Hat2 and Mis18, the CENP-A chaperone Scm3HJURP exhibits similar temporal dissociation–reassociation at centromeres during mitosis [9,12]. Thus, our analyses identify Eic1 and Eic2 as two additional proteins that are released from centromeres in early mitosis and reloaded on centromeres in mid-anaphase. The finding that Eic1 and Eic2 exhibit similar association dynamics as Mis18 through the cell cycle is consistent with our identification of Eic1 and Eic2 being tightly associated with Mis18.Figure 2.


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 associate specifically with centromeres. (a) Eic1 and Eic2 bind the central domain of S. pombe centromeres. qChIP analyses showing enrichments of GFP-tagged Eic1 and Eic2 at the central cores (cc) of centromeres 1, 2 and 3 and imr repeats of centromere 1, relative to the act1 locus. Error bars represent standard deviation between at least three biological replicates. (b) Eic1 and Eic2 exhibit very similar genome-wide association profiles as Mis18 and Scm3HJURP. A comparison between the ChIP-seq profiles of GFP-tagged Eic1, Eic2, Mis18 and Scm3 across centromere 2 is presented, alongside a schematic diagram of centromere 2 (bottom). Normalized coverage represents the number of sequencing fragments obtained from anti-GFP IP normalized to that obtained from the input. (c) Eic1 and Eic2 exhibit very similar cell-cycle localization dynamics as Mis18 and Scm3HJURP. Immunofluorescence of S. pombe cells expressing GFP-tagged Eic1 or Eic2 stained with antibodies to GFP (green) and Cnp1CENP-A (red), and DAPI (blue). Both Eic1 and Eic2 dissociate from centromeres during prometaphase to mid-anaphase of mitosis ((iii)–(vii)) and subsequently reassociate. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140043F2: Eic1 and Eic2 associate specifically with centromeres. (a) Eic1 and Eic2 bind the central domain of S. pombe centromeres. qChIP analyses showing enrichments of GFP-tagged Eic1 and Eic2 at the central cores (cc) of centromeres 1, 2 and 3 and imr repeats of centromere 1, relative to the act1 locus. Error bars represent standard deviation between at least three biological replicates. (b) Eic1 and Eic2 exhibit very similar genome-wide association profiles as Mis18 and Scm3HJURP. A comparison between the ChIP-seq profiles of GFP-tagged Eic1, Eic2, Mis18 and Scm3 across centromere 2 is presented, alongside a schematic diagram of centromere 2 (bottom). Normalized coverage represents the number of sequencing fragments obtained from anti-GFP IP normalized to that obtained from the input. (c) Eic1 and Eic2 exhibit very similar cell-cycle localization dynamics as Mis18 and Scm3HJURP. Immunofluorescence of S. pombe cells expressing GFP-tagged Eic1 or Eic2 stained with antibodies to GFP (green) and Cnp1CENP-A (red), and DAPI (blue). Both Eic1 and Eic2 dissociate from centromeres during prometaphase to mid-anaphase of mitosis ((iii)–(vii)) and subsequently reassociate. Scale bar, 5 μm.
Mentions: CENP-A and all known fission yeast kinetochore proteins localize specifically at centromeres and are enriched over the central domain region of centromeres. Both Eic1 and Eic2 associate with Mis18, which is known to localize to fission yeast centromeres for most of the cell cycle, apart from early prophase to mid-anaphase of mitosis [13]. To examine the localization of Eic1 and Eic2, the endogenous genes expressing Eic1 and Eic2 were fused to GFP. Quantitative chromatin immunoprecipitation (qChIP) analyses demonstrated that, as with Mis18 and other kinetochore proteins, both Eic1-GFP and Eic2-GFP are enriched over the central kinetochore domain (central core plus imr repeats) of fission yeast centromeres (figure 2a). Genome-wide ChIP-seq analyses confirmed this finding and also demonstrated that Eic1-GFP and Eic2-GFP, much like Mis18-GFP and Scm3-GFP, are undetectable at other regions of the genome including the heterochromatic outer repeats of centromeres (figure 2b and electronic supplementary material, figure S2). Immunostaining revealed that both Eic1 and Eic2 GFP-tagged proteins co-localize with Cnp1CENP-A at clustered centromeres in interphase cells (figure 2c(i),(ii)). However, as with Mis16RbAp46/48/Hat2 and Mis18 [11,13], Eic1-GFP and Eic2-GFP dissociate from centromeres in early mitosis (prometaphase), just as the centromeres (Cnp1) begin to form two separate clusters as they biorient on the spindle (figure 2c(iii),(iv)). Moreover, both Eic1-GFP and Eic2-GFP reassociate with centromeres in mid-anaphase when centromeres (Cnp1) and chromosomes (DAPI) have clearly segregated to opposite spindle poles (figure 2c(viii),(ix)). Apart from Mis16RbAp46/48/Hat2 and Mis18, the CENP-A chaperone Scm3HJURP exhibits similar temporal dissociation–reassociation at centromeres during mitosis [9,12]. Thus, our analyses identify Eic1 and Eic2 as two additional proteins that are released from centromeres in early mitosis and reloaded on centromeres in mid-anaphase. The finding that Eic1 and Eic2 exhibit similar association dynamics as Mis18 through the cell cycle is consistent with our identification of Eic1 and Eic2 being tightly associated with Mis18.Figure 2.

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