Centromere Protein A Nucleosomes Research Articles

The cryo-EM structure of the CENP-A nucleosome in complex with ggKNL2.

Centromere protein A (CENP-A) nucleosomes containing the centromere-specific histone H3 variant CENP-A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP-A deposition via the CENP-A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP-C-like motif, and our previous study suggested that ggKNL2 directly binds to the CENP-A nucleosome to recruit HJURP/CENP-A to the centromere. However, the molecular basis for CENP-A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment containing the CENP-C-like motif. Chicken KNL2 distinguishes between CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the C-terminal tail and the RG-loop of CENP-A are simultaneously recognized as CENP-A characteristics. The CENP-A nucleosome-ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.

The function, structure and dynamic organization of centromeres and kinetochores

It is a fundamental task to ensure the faithful transmission of genetic information across generations for eukaryote species. The centromere is a specialized chromosomal region that is essential for mediating sister chromatid alignment and separation during mitosis. Centromere identity is epigenetically determined by nucleosome-containing centromere protein A (CENP-A). The CENP-A nucleosome provides the foundation for the association of the inner kinetochore and the assembly of the outer kinetochore in mitosis. Here we review centromere identity determination, inner kinetochore function and assembly, and outer kinetochore function and assembly. In particular, we focus on the recent advances in the structure-activity relationship of the constitutive centromere-associated network (CCAN). CCAN structure information sheds new light on our understanding of centromere and kinetochore functions and dynamic organization.

Centromere Identity and the Regulation of Chromosome Segregation.

Eukaryotes segregate their chromosomes during mitosis and meiosis by attaching chromosomes to the microtubules of the spindle so that they can be distributed into daughter cells. The complexity of centromeres ranges from the point centromeres of yeast that attach to a single microtubule to the more complex regional centromeres found in many metazoans or holocentric centromeres of some nematodes, arthropods and plants, that bind to dozens of microtubules per kinetochore. In vertebrates, the centromere is defined by a centromere specific histone variant termed Centromere Protein A (CENP-A) that replaces histone H3 in a subset of centromeric nucleosomes. These CENP-A nucleosomes are distributed on long stretches of highly repetitive DNA and interspersed with histone H3 containing nucleosomes. The mechanisms by which cells control the number and position of CENP-A nucleosomes is unknown but likely important for the organization of centromeric chromatin in mitosis so that the kinetochore is properly oriented for microtubule capture. CENP-A chromatin is epigenetically determined thus cells must correct errors in CENP-A organization to prevent centromere dysfunction and chromosome loss. Recent improvements in sequencing complex centromeres have paved the way for defining the organization of CENP-A nucleosomes in centromeres. Here we discuss the importance and challenges in understanding CENP-A organization and highlight new discoveries and advances enabled by recent improvements in the human genome assembly.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome.

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

Quiescent Cells Actively Replenish CENP-A Nucleosomes to Maintain Centromere Identity and Proliferative Potential.

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)2 tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.

Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell.

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

Centromere inheritance through the germline.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.

Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the ‘arginine anchor’ on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.

Orchestrating the Specific Assembly of Centromeric Nucleosomes.

Centromeres are chromosomal loci that are defined epigenetically in most eukaryotes by incorporation of a centromere-specific nucleosome in which the canonical histone H3 variant is replaced by Centromere Protein A (CENP-A). Therefore, the assembly and propagation of centromeric nucleosomes are critical for maintaining centromere identify and ensuring genomic stability. Centromeres direct chromosome segregation (during mitosis and meiosis) by recruiting the constitutive centromere-associated network of proteins throughout the cell cycle that in turn recruits the kinetochore during mitosis. Assembly of centromere-specific nucleosomes in humans requires the dedicated CENP-A chaperone HJURP, and the Mis18 complex to couple the deposition of new CENP-A to the site of the pre-existing centromere, which is essential for maintaining centromere identity. Human CENP-A deposition occurs specifically in early G1, into pre-existing chromatin, and several additional chromatin-associated complexes regulate CENP-A nucleosome deposition and stability. Here we review the current knowledge on how new CENP-A nucleosomes are assembled selectively at the existing centromere in different species and how this process is controlled to ensure stable epigenetic inheritance of the centromere.

Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere.

Inheritance of each chromosome depends upon its centromere. A histone H3 variant, centromere protein A (CENP-A), is essential for epigenetically marking centromere location. We find that CENP-A is quantitatively retained at the centromere upon which it is initially assembled. CENP-C binds to CENP-A nucleosomes and is a prime candidate to stabilize centromeric chromatin. Using purified components, we find that CENP-C reshapes the octameric histone core of CENP-A nucleosomes, rigidifies both surface and internal nucleosome structure, and modulates terminal DNA to match the loose wrap that is found on native CENP-A nucleosomes at functional human centromeres. Thus, CENP-C affects nucleosome shape and dynamics in a manner analogous to allosteric regulation of enzymes. CENP-C depletion leads to rapid removal of CENP-A from centromeres, indicating their collaboration in maintaining centromere identity.

Torque Mediated Kinetics of CENP-A Nucleosomes Reveals Resilience to Tension

In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes. In the centromere, the region of the chromosome that links sister chromatids, histone H3 is replaced by CENP-A (Centromere protein A). Since CENP-A chromatin is the point of contact between the microtubules/kinetochore complex and the rest of the chromosome, these regions endure very high forces. Whereas canonical nucleosomes unwrap at 3pN and disassemble at 15-20pN of force, the estimated forces applied by microtubili are much higher. To investigate how CENP-A nucleosomes respond to externally applied forces and torque, we studied how CENP-A chromatin responds to defined stretching forces and torque generated by magnetic tweezers. With this technique, a single DNA molecule containing a few (up to 10) nucleosomes is tethered between a glass surface and a magnetic bead. By applying stretching forces at constant negative and positive supercoiling, the forces needed for disassembly of canonical H3 and CENP-A nucleosomes can be obtained and compared. By measuring the DNA end-to-end length as a function of applied rotations before and after CENP-A nucleosomes are removed from the DNA, the linking number of the nucleosomes can be determined. Interestingly, while inducing supercoiling at constant low (<0.5pN) forces, unlike canonical nucleosomes, CENP-A nucleosomes appear to respond to the applied torque. By further analyzing the structural stability of CENP-A nucleosomes under stretching force and torque, we hope to unravel the mechanism by which CENP-A nucleosomes resists disassembly during mitosis in vivo.

The Binding Landscapes of the H3/H4 and CENP-A/H4 Dimers

Centromere protein A (CENP-A) is a centromere-specific variant of histone H3 and shares almost 50% amino acid identity to canonical histone H3. CENP-A is required for packaging the centromere, to facilitate separation of sister chromatids during mitosis. Indeed, significant structural similarities have been reported to exist between CENP-A/H4 dimers and H3/H4 dimers in co-crystals. In this work, we used molecular dynamics simulations to map the binding free energy landscape for the CENP-A/H4 and H3/H4 dimers. The Associated memory, Water mediated, Structure and Energy Model (AWSEM) and umbrella sampling constraints were applied for each simulation system towards obtaining two-dimensional free energy profiles of monomeric protein association and folding. Surprisingly, our calculations revealed significant thermodynamic distinctions between dimerization profiles of CENP-A/H4 and of H3/H4 pairs. Furthermore, we also investigated the actions of various histones chaperones, finding that free energy landscapes of the CENP-A/H4 dimer is significantly remodeled in the presence of its cognate chaperone HJURP. The obtained results are in general agreement with the available experimental data and provide new thermodynamic insights into the mechanisms that form the basis of canonical and histone variant CENP-A nucleosomes assembly in vivo.

Posttranslational modification of CENP-A influences the conformation of centromeric chromatin

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.

Dimerization of the CENP-A assembly factor HJURP is required for centromeric nucleosome deposition

The epigenetic mark of the centromere is thought to be a unique centromeric nucleosome that contains the histone H3 variant, centromere protein-A (CENP-A). The deposition of new centromeric nucleosomes requires the CENP-A-specific chromatin assembly factor HJURP (Holliday junction recognition protein). Crystallographic and biochemical data demonstrate that the Scm3-like domain of HJURP binds a single CENP-A-histone H4 heterodimer. However, several lines of evidence suggest that HJURP forms an octameric CENP-A nucleosome. How an octameric CENP-A nucleosome forms from individual CENP-A/histone H4 heterodimers is unknown. Here, we show that HJURP forms a homodimer through its C-terminal domain that includes the second HJURP_C domain. HJURP exists as a dimer in the soluble preassembly complex and at chromatin when new CENP-A is deposited. Dimerization of HJURP is essential for the deposition of new CENP-A nucleosomes. The recruitment of HJURP to centromeres occurs independent of dimerization and CENP-A binding. These data provide a mechanism whereby the CENP-A pre-nucleosomal complex achieves assembly of the octameric CENP-A nucleosome through the dimerization of the CENP-A chaperone HJURP.

The octamer is the major form of CENP-A nucleosomes at human centromeres

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171 bp monomers at normal centromeres, and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA wrapping behavior as octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on their conventional counterparts containing canonical H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

Octameric CENP-A Nucleosomes Are Present at Human Centromeres throughout the Cell Cycle

The presence of a single centromere on each chromosome that signals formation of a mitotic kinetochore is central to accurate chromosome segregation. The histone H3 variant centromere protein-A (CENP-A) is critical for centromere identity and function; CENP-A chromatin acts as an epigenetic mark to direct both centromere and kinetochore assembly. Interpreting the centromere epigenetic mark ensures propagation of a single centromere per chromosome to maintain ploidy. Thus, understanding the nature of CENP-A chromatin is crucial for all cell divisions. However, there are ongoing debates over the fundamental composition of centromeric chromatin. Here we show that natively assembled human CENP-A nucleosomes are octameric throughout the cell cycle. Using total internal reflection fluorescence (TIRF)-coupled photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells, we find that the majority of CENP-A nucleosomes contain CENP-A dimers. In addition, we detect the presence of H2B and H4 in these nucleosomes. Surprisingly, CENP-A associated with the chaperone HJURP can exist as either monomer or dimer, indicating possible assembly intermediates. Thus, our findings indicate that octameric CENP-A nucleosomes mark the centromeric region to ensure proper epigenetic inheritance and kinetochore assembly.

Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle-restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle-restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.

HJURP Regulates Cellular Senescence in Human Fibroblasts and Endothelial Cells Via a p53-Dependent Pathway

Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

CENP-A Nucleosomes adopt an Unconventional Shape

The centromere is the locus that ensures proper segregation of chromosomes through generations. In all eukaryotes, except for budding yeast, centromere location is maintained by an epigenetic mechanism. The most attractive candidate to provide the centromere-specifying epigenetic mark is the histone variant centromere protein A (CENP-A) that replaces histone H3 in centromeric nucleosomes. The (CENP-A/H4)2 heterotetramer crystal structure (Sekulic et al., 2010, Nature 467:347-351) led to our proposal that CENP-A could distinguish centromeric chromatin from the rest of the chromosome via structural deviation from within the folded octameric core of the nucleosome. Our model is based on the intranucleosomal rigidity conferred by CENP-A and rotation at the CENP-A/CENP-A interface. If preserved upon nucleosome formation the rotation would alter the radius of curvature of nucleosomal DNA and histone subunit packing. In the subsequent crystal structure of the CENP-A nucleosome (Tachiwana et al., 2011, Nature 476:232-235), though, nucleosomal DNA curvature and histone subunit packing are as in conventional nucleosome. To investigate the conformation of CENP-A nucleosomes in solution we used intranucleosomal FRET. We find that CENP-A nucleosomes heavily populate an atypical conformation where H2A/H2B dimers are 4 Å further apart at steady state relative to conventional nucleosomes. These data suggest that CENP-A nucleosomes in solution exist in equilibrium between at least two structural conformations: one that is similar to the conventional nucleosomes and one that is altered by rotation at the CENP-A/CENP-A interface. To further interrogate the structure of CENP-A nucleosomes in solution we are also employing SAXS (small-angle X-ray scattering) and SANS (small-angle neutron scattering). SANS experiments exploit contrast variation schemes to provide information on DNA and protein subunits independently. Our ongoing biophysical studies promise to provide critical insight into how CENP-A nucleosomes distinguish centromeric chromatin in the context of mammalian chromosomes.