Regional Centromeres Contain a Centromeric Histone H3 Variant and Repetitive DNA

KEY CONCEPTS
- Centromeres are characterized by a centromere-specific histone H3 variant and often have heterochromatin that is rich in satellite DNA sequences.
-Installation of the centromere-specific histone H3 is an epigenetic and primary determinant that specifies a functional centromere.
-Centromeres in higher eukaryotic chromosomes contain large amounts of repetitive DNA and unique histone variants.
-The function of the ever-present repetitive centromeric DNA is not known.
The region of the chromosome at which the centromere forms was originally thought to be defined by DNA sequences, yet recent studies in plants, animals, and fungi have shown that centromeres are specified epigenetically by chromatin structure. Centromerespecific histone H3 (known as Cse4 in yeast, CENP-A in higher eukaryotes, and more generically as CenH3 ) appears to be a primary determinant in establishing functional centromeres and kinetochore assembly sites. This finding explains the old puzzle of why specific DNA sequences could not be identified as “the centromeric DNA” and why there is so much variation in centromere-associated DNA sequences among closely related species. FIGURE 1 shows the role of the centromeric histone H3, CENP-A, in organizing the centromere at the point of kinetochore attachment. Several working models of the spatial arrangement of chromatin relative to the kinetochore are shown.

FIGURE 7.25 Organization of CENP-A and H3 Nucleosomes in Centromeres. (a) 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. (d) The boustrophedon model of centromeric CENP-A-containing chromatin was proposed based on super-resolution microscopy.
Data from Fukagawa, T., et al. (2014). Dev Cell 30: 496–508doi: (10.1016/j.devcel.2014.08.016.
Centromeres are highly specialized chromatin structures that occupy the same site for many generations, despite the fact that they can be repositioned without DNA transposition. In eukaryotic chromosomes, the centromere-specific histone H3 variant CenH3 replaces the normal H3 histone at sites where centromeres reside and kinetochores attach chromosomes to spindle fibers. This specialized centromeric chromatin is the foundation for binding of other centromere-associated proteins. In addition, other histones at the centromere (including H2A and canonical H3) are subject to posttranslational modifications that are required for normal binding of centromeric proteins and accurate chromosome segregation, indicating that the epigenetic pattern that defines a centromere is complex. This view represents a paradigm shift in how we understand centromere formation, identity, and function. CenH3 is a nucleosomal protein and not a DNA sequence per se; thus, the centromere is now regarded as being primarily epigenetic in its specification. The role of satellite DNA sequences, which are also characteristic of centromeres, remains difficult to ascertain, despite their prevalence and conservation. Research has now turned to understanding the role of nucleosome assembly factors that are specific to CenH3 installation. New questions address matters of specificity, such as how do cells maintain a uniform level of CenH3 at centromeres following replication?
The length of DNA required for centromeric function is often quite long. The short, discrete elements of Saccharomyces cerevisiae appear to be an exception to the general rule. S. cerevisiae is the only case so far in which centromeric DNA can be identified by its ability to confer stability on plasmids. A related approach has been used with the yeast Schizosaccharomyces pombe. S. pombe has only three chromosomes, and the region containing each centromere has been identified by deleting most of the sequences of each chromosome to create a stable minichromosome. This approach locates the centromeres within regions of 40 to 100 kb that consist largely or entirely of repetitious DNA. Attempts to localize centromeric functions in Drosophila chromosomes suggest that they are dispersed in a large region of 200 to 600 kb. The large size of this type of centromere may reflect multiple specialized functions, including kinetochore assembly and sister chromatid pairing.
The size of the centromere in Arabidopsis is comparable. Each of the five chromosomes has a centromeric region in which recombination is very largely suppressed. This region occupies >500 kb. The primary motif comprising the heterochromatin of primate centromeres is the α-satellite DNA, which consists of tandem arrays of a 171-bp repeating unit . There is significant variation between individual repeats, although those at any centromere tend to be better related to one another than to members of the family in other locations.
Current models for regional centromere organization and function invoke alternating chromatin domains, with clusters of CenH3 nucleosomes interspersed among clusters of nucleosomes with H3 and some of the histone variant H2A.Z. Different histones are subject to centromere-specific patterns of modification. The CenH3 nucleosomes form the chromatin foundation for recruitment and assembly of the other proteins that eventually comprise a functional kinetochore. The formation of neocentromeres that contain CenH3 but not α-satellite DNA provide important evidence for the idea of centromeres being epigenetically determined. Key questions remain as to the role of repetitive DNA and alternating chromatin domains in forming the large bipartite kinetochore structure on replicated sister centromeres.