Propagation of centromeric chromatin requires exit from mitosis

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.

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Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

The Journal of Cell Biology ◽

10.1083/jcb.200804078 ◽

2008 ◽

Vol 183 (7) ◽

pp. 1193-1202 ◽

Cited By ~ 40

Author(s):

Owen J. Marshall ◽

Alan T. Marshall ◽

K.H. Andy Choo

Keyword(s):

Chromatin Immunoprecipitation ◽

Protein A ◽

Three Dimensional ◽

Higher Order ◽

Order Structure ◽

Mitotic Chromosomes ◽

Centromeric Chromatin ◽

Centromere Protein A ◽

Transmission Electron ◽

Histone H3 Variant

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.

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Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

The Journal of Cell Biology ◽

10.1083/jcb.200701065 ◽

2007 ◽

Vol 176 (6) ◽

pp. 757-763 ◽

Cited By ~ 151

Author(s):

Paul S. Maddox ◽

Francie Hyndman ◽

Joost Monen ◽

Karen Oegema ◽

Arshad Desai

Keyword(s):

Functional Genomics ◽

Protein A ◽

Common Mechanism ◽

Centromeric Chromatin ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Close Proximity ◽

Single Protein ◽

Histone H3 Variant ◽

New Protein

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

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Trans-generational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

10.1101/2020.10.05.325985 ◽

2020 ◽

Author(s):

Reinier F. Prosée ◽

Joanna M. Wenda ◽

Caroline Gabus ◽

Kamila Delaney ◽

Francoise Schwager ◽

...

Keyword(s):

De Novo ◽

Germ Line ◽

Protein A ◽

Histone H3 ◽

Centromeric Chromatin ◽

C Elegans ◽

Centromere Function ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Meiosis I

AbstractCentromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is established de novo on chromatin during diplotene of meiosis I. Here we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but dispensable for centromere maintenance during embryogenesis. Worms homozygous for a CENP-A tail deletion maintain a functional centromere during development, but give rise to inviable offspring because they fail to re-establish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2, and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

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Centromeric chromatin gets loaded

The Journal of Cell Biology ◽

10.1083/jcb.200702020 ◽

2007 ◽

Vol 176 (6) ◽

pp. 735-736 ◽

Cited By ~ 3

Author(s):

Christopher W. Carroll ◽

Aaron F. Straight

Keyword(s):

Chromosome Segregation ◽

Molecular Mechanisms ◽

Protein A ◽

Histone H3 ◽

Chromatin Assembly ◽

Centromeric Chromatin ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Specific Loading ◽

Histone H3 Variant

Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.

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Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

PLoS Biology ◽

10.1371/journal.pbio.3000968 ◽

2021 ◽

Vol 19 (7) ◽

pp. e3000968

Author(s):

Reinier F. Prosée ◽

Joanna M. Wenda ◽

Isa Özdemir ◽

Caroline Gabus ◽

Kamila Delaney ◽

...

Keyword(s):

De Novo ◽

Germ Line ◽

Protein A ◽

Histone H3 ◽

Transgenerational Inheritance ◽

Centromeric Chromatin ◽

C Elegans ◽

Centromere Function ◽

Centromere Protein A ◽

Histone H3 Variant

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

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Diaphanous formin mDia2 regulates CENP-A levels at centromeres

The Journal of Cell Biology ◽

10.1083/jcb.201512034 ◽

2016 ◽

Vol 213 (4) ◽

pp. 415-424 ◽

Cited By ~ 14

Author(s):

Chenshu Liu ◽

Yinghui Mao

Keyword(s):

Quantitative Imaging ◽

Active Form ◽

Protein A ◽

Small Gtpase ◽

Genome Replication ◽

Rac Gtpase ◽

Epigenetic Marker ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Recognition Protein

Centromeres of higher eukaryotes are epigenetically defined by centromere protein A (CENP-A), a centromere-specific histone H3 variant. The incorporation of new CENP-A into centromeres to maintain the epigenetic marker after genome replication in S phase occurs in G1 phase; however, how new CENP-A is loaded and stabilized remains poorly understood. Here, we identify the formin mDia2 as essential for stable replenishment of new CENP-A at centromeres. Quantitative imaging, pulse-chase analysis, and high-resolution ratiometric live-cell studies demonstrate that mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres. Depletion of mDia2 results in a prolonged centromere association of holiday junction recognition protein (HJURP), the chaperone required for CENP-A loading. A constitutively active form of mDia2 rescues the defect in new CENP-A loading caused by depletion of male germ cell Rac GTPase-activating protein (MgcRacGAP), a component of the small GTPase pathway essential for CENP-A maintenance. Thus, the formin mDia2 functions downstream of the MgcRacGAP-dependent pathway in regulating assembly of new CENP-A containing nucleosomes at centromeres.

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Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

The Journal of Cell Biology ◽

10.1083/jcb.201608083 ◽

2017 ◽

Vol 216 (3) ◽

pp. 607-621 ◽

Cited By ~ 33

Author(s):

Yael Nechemia-Arbely ◽

Daniele Fachinetti ◽

Karen H. Miga ◽

Nikolina Sekulic ◽

Gautam V. Soni ◽

...

Keyword(s):

Cell Cycle ◽

Protein A ◽

Epigenetic Mark ◽

Specific Cell ◽

Entry And Exit ◽

Satellite Repeat ◽

Reference Models ◽

Centromere Protein A ◽

Cell Cycle Phases ◽

Dna Unwrapping

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat–containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1–containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2–4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.

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Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin and the associated centromere complex

10.1101/620088 ◽

2019 ◽

Author(s):

Sreyoshi Mitra ◽

Dani L. Bodor ◽

Ana F. David ◽

João F. Mata ◽

Beate Neumann ◽

...

Keyword(s):

Cell Cycle ◽

Genetic Screening ◽

Chromatin Modification ◽

Histone H3 ◽

Centromeric Chromatin ◽

Sumo Protease ◽

Chromatin Regulators ◽

Histone H3 Variant ◽

Key Factor ◽

Labeling Approach

AbstractCentromeres are defined by a unique self-propagating chromatin structure featuring nucleosomes containing the histone H3 variant CENP-A. CENP-A turns over slower than general chromatin and a key question is whether this unusual stability is intrinsic to CENP-A nucleosomes or rather imposed by external factors. We designed a specific genetic screen to identify proteins involved in CENP-A stability based on SNAP-tag pulse chase labeling. Using a double pulse-labeling approach we simultaneously assay for factors with selective roles in CENP-A chromatin assembly. We discover a series of new proteins involved in CENP-A propagation, including proteins with known roles in DNA replication, repair and chromatin modification and transcription, revealing that a broad set of chromatin regulators impacts in CENP-A transmission through the cell cycle. The key factor we find to strongly affect CENP-A stability is SENP6. This SUMO-protease controls not only the levels of chromatin bound CENP-A but is required for the maintenance of virtually the entire centromere and kinetochore, with the exception of CENP-B. Acute depletion of SENP6 protein reveals its requirement for maintaining centromeric CENP-A levels throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex.

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Identification and characterization of centromeric sequences in Xenopus laevis

10.1101/2020.06.23.167643 ◽

2020 ◽

Cited By ~ 1

Author(s):

Owen K Smith ◽

Charles Limouse ◽

Kelsey A Fryer ◽

Nicole A Teran ◽

Kousik Sundararajan ◽

...

Keyword(s):

Xenopus Laevis ◽

Repetitive Sequences ◽

Protein A ◽

Repeat Sequences ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Egg Extracts ◽

Genomic Studies

AbstractCentromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

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CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

The Journal of Cell Biology ◽

10.1083/jcb.201106079 ◽

2011 ◽

Vol 194 (6) ◽

pp. 855-871 ◽

Cited By ~ 134

Author(s):

Ben Moree ◽

Corey B. Meyer ◽

Colin J. Fuller ◽

Aaron F. Straight

Keyword(s):

Chromosome Segregation ◽

Protein A ◽

Histone H3 ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Chromosomal Locus ◽

C Protein ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Complex Protein

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

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