Centromeres of higher eukaryotes are epigenetically maintained; however, the mechanism that underlies centromere inheritance is unknown. Centromere identity and inheritance require the assembly of nucleosomes containing the CenH3 histone variant in place of canonical H3. Although H3 nucleosomes wrap DNA in a left-handed manner and induce negative supercoils, we show here that CenH3 nucleosomes reconstituted from Drosophila histones induce positive supercoils. Furthermore, we show that CenH3 likewise induces positive supercoils in functional centromeres in vivo, using a budding yeast minichromosome system and temperature-sensitive mutations in kinetochore proteins. The right-handed wrapping of DNA around the histone core implied by positive supercoiling indicates that centromere nucleosomes are unlikely to be octameric and that the exposed surfaces holding the nucleosome together would be available for kinetochore protein recruitment. The mutual incompatibility of nucleosomes with opposite topologies could explain how centromeres are efficiently maintained as unique loci on chromosomes.

No abstract

Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, we have biochemically purified a soluble assembly complex that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription.chromosome segregation ͉ Drosophila ͉ histone variant ͉ nucleosome assembly T he centromere is a defining feature of every eukaryotic chromosome, consisting of the unique site that is required for segregation at mitosis and meiosis. Large protein complexes assemble onto centromeres to mediate the attachment of microtubules that pull sister chromatids to opposite poles (1). During every cell cycle, and between meiotic generations, the location of the centromere on each chromosome remains invariant, and centromere positions are faithfully inherited over evolutionary time. This extraordinarily efficient mechanism of centromere maintenance does not seem to depend on specific DNA sequences found at centromeres. For example, although higher eukaryotic centromeres are embedded in multimegabase arrays of tandemly repetitive satellite sequences (2), in rare cases, functionally normal centromeres are found in regions that lack any common sequence features (3, 4).Despite the sequence heterogeneity of centromeric DNA, centromeres are universally marked by the presence of a centromere-specific histone 3 variant [centromeric histone 3 (CenH3)] (5). Most of the DNA in a cell is packaged into nucleosomes in which Ϸ147 bp of DNA is wrapped around histones H3͞H4͞H2A͞H2B. But at centromeres, CenH3 replaces canonical histone H3 (6, 7) and is essential for attachment to spindle microtubules. Most eukaryotic cells contain three distinct types of histone 3 variants (8), whose deposition leads to chromatin differentiation. Canonical histone H3 is sy...