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

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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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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Propagation of centromeric chromatin requires exit from mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200701066 ◽

2007 ◽

Vol 176 (6) ◽

pp. 795-805 ◽

Cited By ~ 407

Author(s):

Lars E.T. Jansen ◽

Ben E. Black ◽

Daniel R. Foltz ◽

Don W. Cleveland

Keyword(s):

Protein A ◽

S Phase ◽

Epigenetic Mark ◽

Multiprotein Complex ◽

Centromeric Chromatin ◽

Microtubule Attachment ◽

Centromere Protein A ◽

Histone H3 Variant ◽

Multiple Cell ◽

Labeling Approach

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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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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The centromere-specific histone variant Cse4p (CENP-A) is essential for functional chromatin architecture at the yeast 2-μm circle partitioning locus and promotes equal plasmid segregation

The Journal of Cell Biology ◽

10.1083/jcb.200603042 ◽

2006 ◽

Vol 174 (6) ◽

pp. 779-790 ◽

Cited By ~ 45

Author(s):

Sujata Hajra ◽

Santanu Kumar Ghosh ◽

Makkuni Jayaram

Keyword(s):

Chromosome Segregation ◽

Protein A ◽

Histone Variant ◽

Centromeric Chromatin ◽

Plasmid Segregation ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Spindle Disassembly ◽

Late Telophase ◽

2 Μm Plasmid

The centromere protein A homologue Cse4p is required for kinetochore assembly and faithful chromosome segregation in Saccharomyces cerevisiae. It has been regarded as the exquisite hallmark of centromeric chromatin. We demonstrate that Cse4 resides at the partitioning locus STB of the 2-μm plasmid. Cse4p-STB association is absolutely dependent on the plasmid partitioning proteins Rep1p and Rep2p and the integrity of the mitotic spindle. The kinetochore mutation ndc10-1 excludes Cse4p from centromeres without dislodging it from STB. Cse4p-STB association lasts from G1/S through late telophase during the cell cycle. The release of Cse4p from STB chromatin is likely mediated through spindle disassembly. A lack of functional Cse4p disrupts the remodeling of STB chromatin by the RSC2 complex, negates Rep2p binding and cohesin assembly at STB, and causes plasmid missegregation. Poaching of a specific histone variant by the plasmid to mark its partitioning locus with a centromere tag reveals yet another one of the molecular trickeries it performs for achieving chromosome- like fidelity in segregation.

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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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Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A

Journal of Cell Science ◽

10.1242/jcs.114.19.3529 ◽

2001 ◽

Vol 114 (19) ◽

pp. 3529-3542 ◽

Cited By ~ 3

Author(s):

Aaron A. Van Hooser ◽

Ilia I. Ouspenski ◽

Heather C. Gregson ◽

Daniel A. Starr ◽

Tim J. Yen ◽

...

Keyword(s):

Dna Binding Protein ◽

Centromeric Heterochromatin ◽

Additional Mechanism ◽

Centromeric Chromatin ◽

Microtubule Associated Proteins ◽

Kinetochore Assembly ◽

Double Minute ◽

Histone H3 Variant ◽

Associated Proteins ◽

Double Minute Chromosomes

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

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Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat

Molecular Biology of the Cell ◽

10.1091/mbc.e04-09-0788 ◽

2005 ◽

Vol 16 (4) ◽

pp. 1800-1810 ◽

Cited By ~ 29

Author(s):

Nathaniel S. Edwards ◽

Andrew W. Murray

Keyword(s):

Dna Sequences ◽

Cultured Cells ◽

Protein A ◽

Centromeric Dna ◽

Eukaryotic Chromosome ◽

Centromeric Repeat ◽

Kinetochore Assembly ◽

Centromere Protein A ◽

Dna Repeat ◽

Egg Extracts

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.

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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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