Focus on the centre: the role of chromatin on the regulation of centromere identity and function

The centromere is a specialised chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kinetochore, a critical structure that mediates binding of chromosomes to the spindle, monitors bipolar attachment and pulls chromosomes to the poles during anaphase. Identified more than a century ago as the primary constriction of condensed metaphase chromosomes, the centromere remained elusive to molecular characterisation for many years owed to its unusual enrichment in highly repetitive satellite DNA sequences, except in budding yeast. In the last decade, our understanding of centromere structure, organisation and function has increased tremendously. Nowadays, we know that centromere identity is determined epigenetically by the formation of a unique type of chromatin, which is characterised by the presence of the centromere‐specific histone H3 variant CenH3, originally called CENP‐A, which replaces canonical histone H3 at centromeres. CenH3‐chromatin constitutes the physical and functional foundation for kinetochore assembly. This review explores recent studies addressing the structural and functional characterisation of CenH3‐chromatin, its assembly and propagation during mitosis, and its contribution to kinetochore assembly.     

Molecular underpinnings of centromere identity and maintenance

Centromeres direct faithful chromosome inheritance at cell division but are not defined by a conserved DNA sequence. Instead, a specialized form of chromatin containing the histone H3 variant, CENP-A, epigenetically specifies centromere location. We discuss current models where CENP-A serves as the marker for the centromere during the entire cell cycle in addition to generating the foundational chromatin for the kinetochore in mitosis. Recent elegant experiments have indicated that engineered arrays of CENP-A-containing nucleosomes are sufficient to serve as the site of kinetochore formation and for seeding centromeric chromatin that self-propagates through cell generations. Finally, recent structural and dynamic studies of CENP-A-containing histone complexes - before and after assembly into nucleosomes - provide models to explain underlying molecular mechanisms at the centromere.     

HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore

Centromeres of higher eukaryotes are epigenetically marked by the centromere-specific CENP-A nucleosome. New CENP-A recruitment requires the CENP-A histone chaperone HJURP. In this paper, we show that a LacI (Lac repressor) fusion of HJURP drove the stable recruitment of CENP-A to a LacO (Lac operon) array at a noncentromeric locus. Ectopically targeted CENP-A chromatin at the LacO array was sufficient to direct the assembly of a functional centromere as indicated by the recruitment of the constitutive centromere-associated network proteins, the microtubule-binding protein NDC80, and the formation of stable kinetochore–microtubule attachments. An amino-terminal fragment of HJURP was able to assemble CENP-A nucleosomes in vitro, demonstrating that HJURP is a chromatin assembly factor. Furthermore, HJURP recruitment to endogenous centromeres required the Mis18 complex. Together, these data suggest that the role of the Mis18 complex in CENP-A deposition is to recruit HJURP and that the CENP-A nucleosome assembly activity of HJURP is responsible for centromeric chromatin assembly to maintain the epigenetic mark.     

A coordinated interdependent protein circuitry stabilizes the kinetochore ensemble to protect CENP-A in the human pathogenic yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3-5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.     

Pericentric heterochromatin: dynamic organization during early development in mammals

Abstract Constitutive heterochromatin in mammals is essentially found at centromeres, which are key chromosomal elements that ensure proper chromosome segregation. These regions are considered to be epigenetically defined, given that it is not sequence composition but chromatin organization that defines centromere function. How such an epigenetically defined domain, like the centromere, can be established during development and maintained during somatic cell life are fundamental questions. This review discusses the most recent insights into centromeric heterochromatin organization and replication. We further highlight the plasticity of this domain by describing the large-scale re-organization that occurs during development.     

The centromere of budding yeast

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in tethe cell cycle.     

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G₁ phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G₁ phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.     

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.     

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.     

Epigenetic centromere propagation and the nature of CENP-a nucleosomes

Centromeres direct chromosome inheritance, but in multicellular organisms their positions on chromosomes are primarily specified epigenetically rather than by a DNA sequence. The major candidate for the epigenetic mark is chromatin assembled with the histone H3 variant CENP-A. Recent studies offer conflicting evidence for the structure of CENP-A-containing chromatin, including the histone composition and handedness of the DNA wrapped around the histones. We present a model for the assembly and deposition of centromeric nucleosomes that couples these processes to the cell cycle. This model reconciles divergent data for CENP-A-containing nucleosomes and provides a basis for how centromere identity is stably inherited.     

Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

Background  

Mammalian centromere formation is dependent on chromatin that contains centromere protein (CENP)-A, which is the centromere-specific histone H3 variant. Human neocentromeres have acquired CENP-A chromatin epigenetically in ectopic chromosomal locations on low-copy complex DNA. Neocentromeres permit detailed investigation of centromeric chromatin organization that is not possible in the highly repetitive alpha satellite DNA present at endogenous centromeres.     

Ectopic Centromere Nucleation by CENP-A in Fission Yeast

The centromere is a specific chromosomal locus that organizes the assembly of the kinetochore. It plays a fundamental role in accurate chromosome segregation. In most eukaryotic organisms, each chromosome contains a single centromere the position and function of which are epigenetically specified. Occasionally, centromeres form at ectopic loci, which can be detrimental to the cell. However, the mechanisms that protect the cell against ectopic centromeres (neocentromeres) remain poorly understood. Centromere protein-A (CENP-A), a centromere-specific histone 3 (H3) variant, is found in all centromeres and is indispensable for centromere function. Here we report that the overexpression of CENP-A^Cnp1 in fission yeast results in the assembly of CENP-A^Cnp1 at noncentromeric chromatin during mitosis and meiosis. The noncentromeric CENP-A preferentially assembles near heterochromatin and is capable of recruiting kinetochore components. Consistent with this, cells overexpressing CENP-A^Cnp1 exhibit severe chromosome missegregation and spindle microtubule disorganization. In addition, pulse induction of CENP-A^Cnp1 overexpression reveals that ectopic CENP-A chromatin can persist for multiple generations. Intriguingly, ectopic assembly of CENP-A^cnp1 is suppressed by overexpression of histone H3 or H4. Finally, we demonstrate that deletion of the N-terminal domain of CENP-A^cnp1 results in an increase in the number of ectopic CENP-A sites and provide evidence that the N-terminal domain of CENP-A prevents CENP-A assembly at ectopic loci via the ubiquitin-dependent proteolysis. These studies expand our current understanding of how noncentromeric chromatin is protected from mistakenly assembling CENP-A.     

At the right place at the right time: novel CENP-A binding proteins shed light on centromere assembly

Centromeres, the chromosomal loci that form the sites of attachment for spindle microtubules during mitosis, are identified by a unique chromatin structure generated by nucleosomes containing the histone H3 variant CENP-A. The apparent epigenetic mode of centromere inheritance across mitotic and meiotic divisions has generated much interest in how CENP-A assembly occurs and how structurally divergent centromeric nucleosomes can specify the centromere complex. Although a substantial number of proteins have been implicated in centromere assembly, factors that can bind CENP-A specifically and deliver nascent protein to the centromere were, thus far, lacking. Several recent reports on experiments in fission yeast and human cells have now shown significant progress on this problem. Here, we discuss these new developments and their implications for epigenetic centromere inheritance.     

Cdk activity couples epigenetic centromere inheritance to cell cycle progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.     

How to build a centromere: from centromeric and pericentromeric chromatin to kinetochore assembly.

The assembly of the centromere, a specialized region of DNA along with a constitutive protein complex which resides at the primary constriction and is the site of kinetochore formation, has been puzzling biologists for many years. Recent advances in the fields of chromatin, microscopy, and proteomics have shed a new light on this complex and essential process. Here we review recently discovered mechanisms and proteins involved in determining mammalian centromere location and assembly. The centromeric core protein CENP-A, a histone H3 variant, is hypothesized to designate centromere localization by incorporation into centromere-specific nucleosomes and is essential for the formation of a functional kinetochore. It has been found that centromere localization of centromere protein A (CENP-A), and therefore centromere determination, requires proteins involved in histone deacetylation, as well as base excision DNA repair pathways and proteolysis. In addition to the incorporation of CENP-A at the centromere, the formation of heterochromatin through histone methylation and RNA interference is also crucial for centromere formation. The assembly of the centromere and kinetochore is complex and interdependent, involving epigenetics and hierarchical protein-protein interactions.     

Determining centromere identity: cyclical stories and forking paths

The centromere is the genetic locus required for chromosome segregation. It is the site of spindle attachment to the chromosomes and is crucial for the transfer of genetic information between cell and organismal generations. Although the centromere was first recognized more than 120 years ago, little is known about what determines its site(s) of activity, and how it contributes to kinetochore formation and spindle attachment. Recent work in this field has supported the hypothesis that most eukaryotic centromeres are determined epigenetically rather than by primary DNA sequence. Here, we review recent studies that have elucidated the organization and functions of centromeric chromatin, and evaluate present-day models for how centromere identity and propagation are determined.     

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization.     

Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin

Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro. Herein, we demonstrate that after DNA replication, replicated, but not unreplicated, DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells. We suggest that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states.