N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin

Abstract The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

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Subunit interactions and arrangements in the fission yeast Mis16–Mis18–Mis19 complex

Life Science Alliance ◽

10.26508/lsa.201900408 ◽

2019 ◽

Vol 2 (4) ◽

pp. e201900408 ◽

Cited By ~ 1

Author(s):

Melanie Korntner-Vetter ◽

Stéphane Lefèvre ◽

Xiao-Wen Hu ◽

Roger George ◽

Martin R Singleton

Keyword(s):

Cell Cycle ◽

Binding Site ◽

Fission Yeast ◽

Histone H3 ◽

Histone H4 ◽

Subunit Interactions ◽

Centromeric Chromatin ◽

Partner Protein ◽

Histone H3 Variant

Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1CENP-A. Cell cycle–specific deposition of Cnp1 requires the Mis16–Mis18–Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18–Mis19 interface.

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N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

10.1101/176206 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kentaro Ohkuni ◽

Reuben Levy-Myers ◽

Jack Warren ◽

Wei-Chun Au ◽

Yoshimitsu Takahashi ◽

...

Keyword(s):

Genome Stability ◽

Histone H3 ◽

Physiological Role ◽

Centromeric Chromatin ◽

E3 Ligases ◽

Physiological Conditions ◽

Evolutionarily Conserved ◽

Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

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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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Epigenetic inheritance of centromere identity in a heterologous system

10.1101/560391 ◽

2019 ◽

Cited By ~ 2

Author(s):

Virginie Roure ◽

Bethan Medina-Pritchard ◽

Eduard Anselm ◽

A. Arockia Jeyaprakash ◽

Patrick Heun

Keyword(s):

Chromosome Segregation ◽

Chromosomal Region ◽

Histone H3 ◽

Epigenetic Inheritance ◽

Centromeric Chromatin ◽

Centromere Proteins ◽

Heterologous System ◽

Histone H3 Variant ◽

Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

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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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CENP-H–containing Complex Facilitates Centromere Deposition of CENP-A in Cooperation with FACT and CHD1

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0065 ◽

2009 ◽

Vol 20 (18) ◽

pp. 3986-3995 ◽

Cited By ~ 91

Author(s):

Masahiro Okada ◽

Katsuya Okawa ◽

Toshiaki Isobe ◽

Tatsuo Fukagawa

Keyword(s):

Chromatin Remodeling ◽

Mutant Cell ◽

Histone H3 ◽

Dependent Manner ◽

Centromeric Chromatin ◽

Conditional Mutant ◽

Histone H3 Variant ◽

A Subunit

Centromere identity is thought to be determined by epigenetic mechanisms. The centromere-specific histone H3 variant CENP-A plays a central role in specifying the locus where the centromere is constructed. However, the precise mechanisms that target CENP-A to centromeric chromatin are poorly understood. Here, we show that facilitates chromatin transcription (FACT) localizes to centromeres in a CENP-H–containing complex-dependent manner. In conditional mutant cell lines for SSRP1, a subunit of FACT, centromere targeting of newly synthesized CENP-A is severely inhibited. The chromatin remodeling factor CHD1 binds to SSRP1 both in vivo and in vitro and associates with centromeres. The centromeric localization of CHD1 is lost in SSRP1-depleted cells. RNA interference knockdown of CHD1 leads to a decrease in the amount of centromere localized CENP-A. These findings indicate that the CENP-H–containing complex facilitates deposition of newly synthesized CENP-A into centromeric chromatin in cooperation with FACT and CHD1.

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The N-terminal Tail of C. elegans CENP-A Interacts with KNL-2 and is Essential for Centromeric Chromatin Assembly

10.1101/2020.12.28.424576 ◽

2020 ◽

Author(s):

Christian de Groot ◽

Jack Houston ◽

Bethany Davis ◽

Adina Gerson-Gurwitz ◽

Joost Monen ◽

...

Keyword(s):

Histone H3 ◽

Direct Interaction ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Centromeric Chromatin ◽

C Elegans ◽

Histone Fold ◽

Kinetochore Assembly ◽

Histone H3 Variant ◽

Evolutionary Variation

ABSTRACTCentromeres are epigenetically defined by the presence of the centromere-specific histone H3 variant CENP-A. A specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, which rely on a CENP-A-dependent centromere. Here, we show that the extended N-terminal tail of C. elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly. Removal of this region of the CENP-A N-Tail prevents loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-Tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-Tail containing the predicted structured region binds to KNL-2, a conserved SANTA and Myb domain-containing protein (referred to as M18BP1 in vertebrates), that is specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode C. briggsae, despite divergence of the N-Tail and KNL-2 primary sequences. Thus, the extended N-Tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates an interaction of the specialized histone fold of CENP-A with KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.

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The ratio between centromeric proteins CENP-A and CENP-C maintains homeostasis of human centromeres

10.1101/604223 ◽

2019 ◽

Cited By ~ 4

Author(s):

Daniël P. Melters ◽

Tatini Rakshit ◽

Minh Bui ◽

Sergei A. Grigoryev ◽

David Sturgill ◽

...

Keyword(s):

Mitotic Spindle ◽

De Novo ◽

Histone H3 ◽

Chromosomal Locus ◽

Centromeric Chromatin ◽

Dna Accessibility ◽

Nucleosome Dynamics ◽

Histone H3 Variant ◽

Correct Ratio

AbstractThe centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

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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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