Centromeric Chromatin Research Articles

Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration.

Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.

Sequence Composition Underlying Centromeric and Heterochromatic Genome Compartments of the Pacific Oyster Crassostrea gigas.

Segments of the genome enriched in repetitive sequences still present a challenge and are omitted in genome assemblies. For that reason, the exact composition of DNA sequences underlying the heterochromatic regions and the active centromeres are still unexplored for many organisms. The centromere is a crucial region of eukaryotic chromosomes responsible for the accurate segregation of genetic material. The typical landmark of centromere chromatin is the rapidly-evolving variant of the histone H3, CenH3, while DNA sequences packed in constitutive heterochromatin are associated with H3K9me3-modified histones. In the Pacific oyster Crassostrea gigas we identified its centromere histone variant, Cg-CenH3, that shows stage-specific distribution in gonadal cells. In order to investigate the DNA composition of genomic regions associated with the two specific chromatin types, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the Cg-CenH3- and H3K9me3-associated sequences. CenH3-associated sequences were assigned to six groups of repetitive elements, while H3K9me3-associated-ones were assigned only to three. Those associated with CenH3 indicate the lack of uniformity in the chromosomal distribution of sequences building the centromeres, being also in the same time dispersed throughout the genome. The heterochromatin of C. gigas exhibited general paucity and limited chromosomal localization as predicted, with H3K9me3-associated sequences being predominantly constituted of DNA transposons.

The centromere comes into focus: from CENP-A nucleosomes to kinetochore connections with the spindle.

Eukaryotic chromosome segregation relies upon specific connections from DNA to the microtubule-based spindle that forms at cell division. The chromosomal locus that directs this process is the centromere, where a structure called the kinetochore forms upon entry into mitosis. Recent crystallography and single-particle electron microscopy have provided unprecedented high-resolution views of the molecular complexes involved in this process. The centromere is epigenetically specified by nucleosomes harbouring a histone H3 variant, CENP-A, and we review recent progress on how it differentiates centromeric chromatin from the rest of the chromosome, the biochemical pathway that mediates its assembly and how two non-histone components of the centromere specifically recognize CENP-A nucleosomes. The core centromeric nucleosome complex (CCNC) is required to recruit a 16-subunit complex termed the constitutive centromere associated network (CCAN), and we highlight recent structures reported of the budding yeast CCAN. Finally, the structures of multiple modular sub-complexes of the kinetochore have been solved at near-atomic resolution, providing insight into how connections are made to the CCAN on one end and to the spindle microtubules on the other. One can now build molecular models from the DNA through to the physical connections to microtubules.

Hec1/Ndc80 Tail Domain Function at the Kinetochore-Microtubule Interface.

Successful mitotic cell division is critically dependent on the formation of correct attachments between chromosomes and spindle microtubules. Microtubule attachments are mediated by kinetochores, which are large proteinaceous structures assembled on centromeric chromatin of mitotic chromosomes. These attachments must be sufficiently stable to transduce force; however, the strength of these attachments are also tightly regulated to ensure timely, error-free progression through mitosis. The highly conserved, kinetochore-associated NDC80 complex is a core component of the kinetochore-microtubule attachment machinery in eukaryotic cells. A small, disordered region within the Hec1 subunit of the NDC80 complex – the N-terminal “tail” domain – has been actively investigated during the last decade due to its roles in generating and regulating kinetochore-microtubule attachments. In this review, we discuss the role of the NDC80 complex, and specifically the Hec1 tail domain, at the kinetochore-microtubule interface, and how recent studies provide a more unified view of Hec1 tail domain function.

Epigenetic regulation of centromere function.

The centromere is a specialized region on the chromosome that directs equal chromosome segregation. Centromeres are usually not defined by DNA sequences alone. How centromere formation and function are determined by epigenetics is still not fully understood. Active centromeres are often marked by the presence of centromeric-specific histone H3 variant, centromere protein A (CENP-A). How CENP-A is assembled into the centromeric chromatin during the cell cycle and propagated to the next cell cycle or the next generation to maintain the centromere function has been intensively investigated. In this review, we summarize current understanding of how post-translational modifications of CENP-A and other centromere proteins, centromeric and pericentric histone modifications, non-coding transcription and transcripts contribute to centromere function, and discuss their intricate relationships and potential feedback mechanisms.

A Balance Between Elastic and Rigidified CENP-A Nucleosomes Govern Centromeric Chromatin Fidelity

A Balance Between Elastic and Rigidified CENP-A Nucleosomes Govern Centromeric Chromatin Fidelity

Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize.

In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization.

A Different Twist on Centromeric Chromatin

Nucleosomes at the centromere contain CENP-A, a histone H3 variant that establishes a specific, yet poorly defined centromeric chromatin architecture. In this issue of Structure, Takizawa etal. (2019) describe an untwisted configuration for an H3-CENP-A-H3 tri-nucleosome that mimics centromeric chromatin. Untwisting may increase centromeric-protein accessibility to CENP-A in compacted chromatin.

Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome

The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres.

CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization.

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A-CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A-binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A-CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

Meiotic Kinetochores Fragment into Multiple Lobes upon Cohesin Loss in Aging Eggs.

SummaryChromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes, that by assembling onto specialized Cenp-A nucleosomes 1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle 3,4. Whereas the centromeres of vertebrate chromosomes comprise Mb of DNA and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere 7–9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here, we describe the cryo-EM structure of the S. cerevisiae inner kinetochore module - the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN–Cenp-ANuc). The structure explains the inter-dependency of CCAN’s constituent sub-complexes and shows how the ‘Y’-shaped opening of CCAN accommodates Cenp-ANuc to allow specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove present in the Cenp-LN sub-complex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore for linking centromeres to the mitotic spindle for chromosome segregation.

Reconstituting Drosophila Centromere Identity in Human Cells

SummaryThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, centromere protein (CENP)-A, yet how its self-propagation is achieved remains poorly understood. Here, we develop 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 CAL1 to be required for dCENP-C loading onto chromatin in cooperation with 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 factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of Drosophila centromere identity.

The SUMO Isopeptidase SENP6 Functions as a Rheostat of Chromatin Residency in Genome Maintenance and Chromosome Dynamics

Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.

Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading.

Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymeraseII transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

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

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.

Structure of the Human Core Centromeric Nucleosome Complex

Centromeric nucleosomes are at the interface of thechromosome and the kinetochore that connects to spindle microtubules in mitosis. The core centromeric nucleosome complex (CCNC) harbors the histone H3 variant, CENP-A, and its binding proteins, CENP-C (through its central domain; CD) and CENP-N (through its N-terminal domain; NT). CENP-C can engage nucleosomes through two domains: the CD and the CENP-C motif (CM). CENP-CCD is part of the CCNC by virtue of its high specificity for CENP-A nucleosomes and ability to stabilize CENP-A at the centromere. CENP-CCM is thought to engage a neighboring nucleosome, either one containing conventional H3 or CENP-A, and a crystal structure of a nucleosome complex containing two copies of CENP-CCM was reported. Recent structures containing a single copy of CENP-NNT bound to the CENP-A nucleosome in the absence of CENP-C were reported. Here, we find that one copy of CENP-N is lost for every two copies of CENP-C on centromeric chromatin just prior to kinetochore formation. We present the structures of symmetric and asymmetric forms of the CCNC that vary in CENP-N stoichiometry. Our structures explain how the central domain of CENP-C achieves its high specificity for CENP-A nucleosomes and how CENP-C and CENP-N sandwich the histone H4 tail. The natural centromeric DNA path in our structures corresponds to symmetric surfaces for CCNC assembly, deviating from what is observed in prior structures using artificial sequences. At mitosis, we propose that CCNC asymmetry accommodates its asymmetric connections at the chromosome/kinetochore interface. VIDEO ABSTRACT.

Cis- and Trans-chromosomal Interactions Define Pericentric Boundaries in the Absence of Conventional Heterochromatin.

The diploid budding yeast Candida albicans harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion, C. albicans can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in C. albicans We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of CEN7 to its peripheral regions. Using publicly available C. albicans high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC). Cis- and trans-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region.