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.2014 Apr 30;4(4):140043.
doi: 10.1098/rsob.140043.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

Affiliations

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly

Lakxmi Subramanian et al. Open Biol..

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.

Keywords: CENP-A; Mis18; centromeres; epigenetics; fission yeast.

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Figures

Figure 1.
Figure 1.
Eic1 and Eic2 are Mis18-interacting proteins. (a) LC-MS/MS analysis of Myc-tagged Mis16RbAp46/48/Hat2 and Mis18 immunoprecipitates fromS. pombe whole cell extracts identifies two previously uncharacterized proteins, Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16). Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Primary sequence alignments of Eic1 (top) and Eic2 (bottom) orthologues identified amongSchizosaccharomyces species. (c) Eic1-GFP co-immunoprecipitates with both Mis16-Myc and Mis18-Myc, while Eic2-GFP only co-immunoprecipitates with Mis18-Myc. The asterisk (*) in the bottom panel denotes the IgG heavy chain. (d) Eic1 directly interacts with Mis18 and Mis16. 6xHis-Eic1 was co-expressed with GST alone, GST-Mis18 or GST-Mis16 inE. coli. Coomassie-stained SDS-PAGE gels showing reciprocal GST (lanes 1–3) and His (lanes 6–8) pulldowns fromE. coli lysates are shown. Also shown are reciprocal pulldowns of 6xHis-Mis16 co-expressed with GST alone or GST-Mis18 (lanes 4–5 and 9–10).
Figure 2.
Figure 2.
Eic1 and Eic2 associate specifically with centromeres. (a) Eic1 and Eic2 bind the central domain ofS. pombe centromeres. qChIP analyses showing enrichments of GFP-tagged Eic1 and Eic2 at the central cores (cc) of centromeres 1, 2 and 3 andimr repeats of centromere 1, relative to theact1 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 ofS. 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.
Figure 3.
Figure 3.
Eic1 is required for Cnp1CENP-A assembly, while Eic2 is dispensable. (a)ts mutations ineic1 affect cell viability, whileeic2Δ cells show no defects in growth. Five-fold serial dilutions of cells spotted on YES + Phloxine B media and incubated at the indicated temperatures; dead cells stain dark pink. (b)eic1 mutants display reduced Cnp1CENP-A levels at centromeres. qChIP analyses of Cnp1CENP-A 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 theact1 locus is presented. (c)eic1 mutants display sensitivity to TBZ, whileeic2Δ cells show no TBZ sensitivity. Five-fold serial dilutions of cells spotted on YES media (untreated) or YES media supplemented with 12.5 μg ml−1 TBZ, and incubated at 25°C. (d)eic2Δ cells display no loss of Cnp1CENP-A at centromeres. qChIP analyses of Cnp1CENP-A association with centromeres in the indicated strains when grown at 32°C. Enrichment of cc2 or cc1/3 DNA relative to theact1 locus is presented. Error bars in (b,d) represent standard deviation between at least three biological replicates.
Figure 4.
Figure 4.
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 theact1 locus is presented in (a–d). Enrichment of cc2 or cc1/3 DNA relative to theact1 locus is presented in (e–h). Error bars represent standard deviation between at least three biological replicates.
Figure 5.
Figure 5.
Analysis of genetic interactions betweeneic1 oreic2 mutants and mutations in Cnp1CENP-A or Cnp1CENP-A assembly factors. (a)eic1+:hygR andeic1-1 cells display reduced growth when combined with mutations inmis18,scm3,cnp1 ormis6. (b)eic2Δ cells display genetic interactions when combined withmis18-262 andscm3-139, but noteic1-1. 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.
Figure 6.
Figure 6.
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 theact1 locus is presented in (a,c,d). Enrichment of cc2 or cc1/3 DNA relative to theact1 locus is presented in (b). Error bars represent standard deviation between at least three biological replicates.
Figure 7.
Figure 7.
Eic1 interacts with three essential subunits of the CCAN/Mis6/Ctf19 complex. (a) LC-MS/MS analysis of GFP-tagged Eic1 or Eic2 immunoprecipitates fromS. pombe whole cell extracts identifies Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 as Eic1-interacting proteins. Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Eic1-GFP co-immunoprecipitates with Fta7-Myc and Cnl2-Myc. In the top panel, the asterisk (*) denotes the IgG heavy chain, and the hash tag (#) denotes a non-specific band.
Figure 8.
Figure 8.
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 theact1 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 withmal2-1. 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 atS. 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 anin 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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