Chromosomes normally have a single centromere, the region where duplicated sister chromatids remain joined until anaphase. In metaphase chromosomes, the centromere is readily apparent as the primary constriction that separates the short and long arms. The centromere is essential for attaching chromosomes to the mitotic spindle and for chromosome segregation during cell division. Abnormal chromosome fragments that lack a centromere (acentric fragments) cannot attach to the spindle and fail to be correctly segregated to the nuclei of either daughter cell.
The centromere is effectively a chromatin structure that specifies where a large multi protein complex, known as a kinetochore, will form on each sister chromatid at later pro phase. The pair of kinetochores serve to tether the centromere to microtubules attached to the spindle poles (see Box 1). At anaphase, the kinetochore microtubules pull the previously paired sister chromatids toward opposite poles of the spindle. The kinetochores control assembly and disassembly of the attached microtubules, which drives chromosome movement.
Box1. COMPONENTS OF THE MITOTIC SPINDLE
In the budding yeast Saccharomyces cerevisiae, the sequences that specify centromere function are very short, as are other functional chromosomal elements (Figure 1). The centromere element (CEN) is about 120–125 bp long and contains three principal sequence elements, of which the central one, CDE II, is particularly important for attaching microtubules to the kinetochore. A centromeric CEN fragment derived from one S. cerevisiae chromosome can replace the centromere of another S. cerevisiae chromosome with no apparent consequence.
Fig1. In S. cerevisiae, chromosome function is dependent on short, defined DNA sequence elements. S. cerevisiae centromeres are very short (often ~120 bp) and, unusually for eukaryotic centromeres, are composed of defined sequence elements. There are three contiguous centromere DNA elements (CDE) of which CDE II and CDE III are the most functionally important. Telomeres are composed of tandem TG-rich repeats. Autonomous replicating sequences are defined by short AT-rich sequences. The three types of short sequence shown here can be combined with foreign DNA to make an artificial chromosome in yeast cells.
S. cerevisiae centromeres are highly unusual because they are very small and the DNA sequences specify the sites of centromere assembly. They do not have counterparts in the centromeres of the fission yeast, S. pombe, or in those of multicellular animals. Centromere size has increased during eukaryote evolution and, in complex organ isms, centromeric DNA is dominated by repeated sequences that evolve rapidly and are species-specific. The relatively rapid evolution of centromeric DNA and associated proteins might contribute to the reproductive isolation of emerging species.
Although centromeric DNA shows remarkable sequence heterogeneity across eukaryotes, centromeres are universally marked by the presence of a centromere- specific variant of histone H3, generically known as CenH3 (the human form of CenH3 is named CENP-A). At centromeres, CenH3/CENP-A replaces the normal histone H3 and is essential for attachment to spindle microtubules. Depending on centromere organization, different numbers of spindle microtubules can be attached (Figure 2).
Fig2. Differences in eukaryotic centromere organization. The budding yeast S. cerevisiae has the simplest form of centromere organization, a point centromere with just ~120 bp of DNA wrapped around a single nucleosome; each kinetochore makes only one stable microtubule attachment during metaphase. As in all centromeres, a centromere-specific variant of histone H3 (generically called CenH3), is implicated in microtubule binding. Other eukaryotes typically have defined regional centromeres that span from tens of kilobases up to a few megabases of DNA, and their kinetochores bind several microtubules. In the fission yeast S. pombe, microtubule attachment is centered on a nonrepetitve core sequence that is flanked by different types of repeat sequence (IMR, innermost; OTR, outermost). In humans, higher-order α-satellite DNA repeats are prominent at centromeres. In addition to binding to nucleosomes containing a CenH3 protein, CENP-A, they also have binding sites for the CENP-B protein. In some eukaryote species, such as the nematode Caenorhabditis elegans, the centromeres are diffuse: multiple kinetochores distributed across the length of the chromosome bind to microtubules of the metaphase spindle. (Chromosomes with diffuse centromeres are said to be holocentric.) Repeat direction is shown by left or right orientation of arrows and chevrons. Variable position/presence of some microtubules and CENP-A homologs is indicated by dashed outer lines. (Adapted from Vagnarelli P et al. [2008] FEBS Lett 582:1950–1959; PMID 18435926. With permission from John Wiley & Sons, Inc. © 2008 Federation of European Biochemical Societies.)
Mammalian centromeres are particularly complex. The associated DNA often extends over several megabases and contains some chromosome-specific as well as repetitive DNA. A major component of human centromeric DNA is α-satellite DNA, whose structure is based on tandem repeats of a 171 bp monomer. Adjacent repeat units can show minor variations in sequence, and occasional tandem amplification of a sequence of several slightly different neighboring repeats results in a higher-order repeat organization. This type of α-satellite DNA is characteristic of centromeres and is marked by 17 bp recognition sites for the centromere-binding protein CENP-B.
Unlike the very small, discrete centromeres of S. cerevisiae, the much larger centromeres of other eukaryotes are not dependent just on sequence organization. Neither specific DNA sequences (for example, α-satellite DNA) nor the DNA binding protein CENP-B are essential or sufficient to dictate the assembly of a functional mammalian centromere. Poorly understood DNA characteristics specify a chromatin conformation that somehow, via epigenetic sequence-independent mechanisms, controls the formation and maintenance of a functional centromere.
