The centromere: chromatin foundation for the kinetochore machinery

Abstract

Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function.

Figure 1

Genomic Organization of Human Centromeres (A) Human centromeres contain α-satellite sequences. In the inner core of the centromere, α-satellite monomers 171 bp long are organized into higher-order repeat (HOR) units, which are amplified, spanning 0.3–5 Mb. Unordered monomer units flank the HOR region. (B) Functional centromeres form on a portion of the HOR, which might be newly evolved and homogenized. Disordered monomer unit sequences in pericentromeres are derived from an ancestral primate centromere. Pericentromere regions are highly heterochromatinized.

Figure 2

Proposed Role of Repetitive Sequences in Centromeres We postulate that the kinetochore location might be able to “drift” on the DNA. If a kinetochore moved over a gene, then it would suppress its expression. This would be deleterious for essential genes. At nonrepetitive centromeres, the probability of this drift affecting a flanking gene is higher than at repetitive centromeres, where the probability of affecting a gene is relatively low. We suggest that repetitive sequences might provide a safety area for centromere “drift.” Pericentromere regions at edge of centromeres might also function as a heterochromatin barrier between centromere and euchromatin regions.

Figure 3

Neocentromere Generation in Various Experimental Systems (A) Isolation of surviving cells after removal of the endogenous centromere on a chromosome by homologous recombination between two LoxP loci flanking the centromere. Surviving cells usually have segregation of the chromosome directed by a neocentromere. (B) Ectopic CENP-A is assembled into a “CENP-A cloud” flanking the endogenous centromere in chicken cells. A CENP-A-rich region might be created by specific 3D packaging of the genome in Candida nuclei. The ectopic CENP-A functions as a site of preferential neocentromere formation in chicken or Candida cells when the endogenous centromere is disrupted. In contrast, neocentromeres are preferentially formed on telomeres in S. pombe.

Figure 4

Use of Human Artificial Chromosomes to Study Centromeric Chromatin A synthetic α-satellite DNA construct was used to generate human artificial chromosomes (HACs) in HT1080 fibrosarcoma cells. An example of the HAC (white arrow) stained for kinetochore protein CENP-C (red) and DNA (blue) is shown. The presence of tetracycline operator sequences in the synthetic array allows targeting of chimeric proteins containing a range of chromatin modification activities into the HAC centromere. These studies reveal that centromere activity requires finely balanced transcription in a repressive environment. Micrograph by Jan Bergmann.

Figure 5

Organization of CENP-A and H3 Nucleosomes in Centromeres (A) Based on ChIP-seq analysis centromeres are ∼40 kb long in chicken, corresponding to 200 nucleosomes per centromere. Of these, 30 are predicted to contain CENP-A (roughly 1 in 6–8 centromeric nucleosomes). Thus, centromeric chromatin is largely composed of nucleosomes containing histone H3. (B and C) The CENP-A chromatin was originally suggested to form an amphipathic organization, with CENP-A on the exterior facing the kinetochore, and H3 largely on the interior. This chromatin was proposed to form either a helix or loop structure. The diagram in (B and C) is based on Blower et al. (2002), but modified to show the lower occupancy of CENP-A nucleosomes in the centromeric chromatin. (D) The boustrophedon model of centromeric CENP-A-containing chromatin was proposed based on super-resolution microscopy (Ribeiro et al., 2010).

Figure 6

The Mis18 Complex Licenses Chromatin for CENP-A Incorporation During replication, CENP-A nucleosomes are divided between the two daughter DNA molecules and are either replaced with H3 (H3.1/H3.3) nucleosomes or leave nucleosome free gaps. During anaphase to telophase, the Mis18 complex localizes to centromeres. Soluble CENP-A-H4 binds to the chaperone HJURP before incorporation, and during early G1 the CENP-A-H4-HJUPRP complex is recruited into the licensed chromatin by binding to the Mis18 complex. Following CENP-A incorporation, H4K20 in CENP-A nucleosomes is monomethylated. This maturation step is required for certain of the constitutive centromere associated network (CCAN) proteins to assemble and subsequently direct assembly of a functional kinetochore.

Figure 7

Molecular Architecture of Kinetochores (A) Molecular architecture of natural kinetochores. Centromere chromatin is crucial for centromere specification and kinetochore assembly at natural centromeres. At the base of the structure are CENP-A containing nucleosomes, centromere specific H3 nucleosomes, and a CENP-T-W-S-X nucleosome-like structure in centromere chromatin. Centromere-specific chromatin structure is established by coordination of these components. CCAN proteins assemble on the centromeric chromatin and the microtubule-binding complex is subsequently recruited to assemble the functional kinetochore. (B) When the CENP-T N terminus or CENP-C N terminus is tethered at a noncentromere locus using the LacI-LacO system, an artificial kinetochore forms on the noncentromeric LacO site. Most centromeric chromatin proteins, including CENP-A, are not detected in the artificial kinetochores. However, the chromosome passenger complex (CPC) and Ndc80 complex are recruited and the artificial kinetochores are fully functional. (C) Summary of studies on creation of artificial kinetochores. Tethering of CENP-T N terminus and/or CENP-C-N terminus can bypass the need for centromere-specific chromatin including CENP-A. CENP-A-mediated artificial kinetochores also have been created in human, chicken, and Drosophila cells and in Xenopus egg extracts.