Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

Samejima, Itaru; Spanos, Christos; Alves, Flávia de Lima; Hori, Tomohide; Perpelescu, Marinela; Zou, Juan; Rappsilber, Juri; Fukagawa, Tatsuo; Earnshaw, William C.

doi:10.1083/jcb.201508072

Cited by 44 publications

(51 citation statements)

References 82 publications

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“…Studies in Saccharomyces cerevisiae report a role for the COMA complex (CENP-O/-P/-Q/-R/-U complex homolog) in the looping of centromere chromatin (45). However, our results plus other recent studies have failed to identify a function for this complex in vertebrate kinetochores (37,41).…”

Section: The Role Of Ccan Proteins In the Maintenance Of Kinetochorecontrasting

confidence: 55%

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Stepwise unfolding supports a subunit model for vertebrate kinetochores

Vargiu

Макаров

Allan

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENP-A-containing nucleosomes into up to five chromatin "subunits"-each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.centromere | kinetochore | CENP-A | centrochromatin | boustrophedon

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Section: The Role Of Ccan Proteins In the Maintenance Of Kinetochorecontrasting

confidence: 55%

“…Indeed, CENP-C is required to efficiently recruit both inner and outer kinetochore components during kinetochore assembly (34)(35)(36)(37).…”

Section: The Role Of Ccan Proteins In the Maintenance Of Kinetochorementioning

confidence: 99%

Stepwise unfolding supports a subunit model for vertebrate kinetochores

Vargiu

Макаров

Allan

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The CENP-S/-X centromere complex, reported to form a heterotetramer with CENP-T/-W (Nishino et al 2012), was not identified as a CENP-W-GFP interactor by our MS analysis. This is consistent with recent reports that predict that CENP-T/-W and CENP-S/-X are part of separate protein cohorts within the kinetochore (Samejima et al 2015). In agreement with previous reports regarding CENP-T/-W interactions, we did not find CenH3 CENP-A in our CENP-W-GFP pull-downs (Hori et al 2008;Abendroth et al 2015), confirming the specificity of our approach.…”

Section: Fact Subunits Interact With the Cenp-t/-w Complexsupporting

confidence: 82%

The CENP-T/-W complex is a binding partner of the histone chaperone FACT

et al. 2016

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The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

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“…CENP-A is extremely stable at centromeres53, thus making it hard to observe large effects. Our detailed proteomic analysis of isolated mitotic chromosomes showed only moderate differences in CENP-A levels even when critical assembly factors such as HJURP or Mis18α were depleted for several days54. Kinetochores depleted of H3K4me2 remained functional for several days until CENP-A levels fell by >50%.…”

Section: Discussionmentioning

confidence: 73%

Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance

et al. 2016

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Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic biology ‘in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoidtetO HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoidtetO array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the ‘epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres.

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Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

Cited by 44 publications

References 82 publications

Stepwise unfolding supports a subunit model for vertebrate kinetochores

Stepwise unfolding supports a subunit model for vertebrate kinetochores

The CENP-T/-W complex is a binding partner of the histone chaperone FACT

Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance

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