The ura4 + gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4 + expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ara4 + gene adjacent to telomeres and at the three centromeric sites tested. The clr4-$5 and rikl-304 mutations cause the most dramatic derepression at two out of three sites within cenl. All six mutations had only slight or intermediate effects on a third site in the center of cenl or on telomeric repression. Strains with lesions at the clr4, rikl, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.[Key Words: S. pombe; PEV; heterochromatin; telomere; mating type] Received October 7, 1994; revised version accepted December 13, 1994.The centromeres of the fission yeast Schizosaccharomyces pombe are complex structures composed of large repetitive arrays symmetrically arranged around a nonrepetitive central domain (Hahnenberger et al. 1991;Murakami et al. 1991;Takahashi et al. 1992;Steiner et al. 1993; cenl is shown in Fig. 1A, below). Long arrays of tandem satellite repeats are frequently associated with the centromeres of many eukaryotes (Miklos and Cotsell 1990;Rattner 1991;Tyler-Smith and Willard 1993). These centromeric satellites are packaged into a highly compacted structure known as constitutive heterochromatin, but it is not known if this contributes to centromere function. It is likely that mammalian satellite repeats contribute to kinetochore formation, but alone, they may not be sufficient to form a functional centromere (Earnshaw et al. 1989;Haaf et al. 1992;Larin et al. 1994).Genes placed in the vicinity of centromeric heterochromatin in mouse cells can exhibit variable states of expression (Butner and Lo 1986). This phenomenon, termed position effect variegation (PEV), was first described in Drosophila, where it was found that genes placed adjacent to centromeric heterochromatin were unstably repressed (Eissenberg 1989;Henikoff 1990;Karpen 1994). In Drosophila, mouse, and human cells, some 1Corresponding author. studies indicate that heterochromatin is often associated with the nuclear periphery (Hochstrasser et al. 1986;Ferguson and Ward 1992;Vourc'h et al. 1993). It is possible that this and other associations serve to lock these regions of chromosomes in an inactive state (Karpen 1994}. In unicellular organisms, such as yeasts, there are chromosomal regions with features characteristic of heterochro...

Histone acetylation may act to mark and maintain transcriptionally active or inactive chromosomal domains through the cell cycle and in different lineages. A novel role for histone acetylation in centromere regulation has been identified. Exposure of fission yeast cells to TSA, a specific inhibitor of histone deacetylase, interferes with repression of marker genes in centromeric heterochromatin, causes chromosome loss, and disrupts the localization of Swi6p, a component of centromeric heterochromatin. Transient TSA treatment induces a heritable hyperacetylated state in centromeric chromatin that is propagated in lineages in the absence of drug. This state is linked in cis to the treated centromere locus and correlates with inheritance of functionally defective centromeres and persistent chromosome segregation problems. Thus, assembly of fully functional centromeres is partly imprinted in the underacetylated or transcriptionally silent state of centromeric chromatin.

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Centromeres attach chromosomes to the spindle during mitosis, thereby ensuring the equal distribution of chromosomes into daughter cells. Transcriptionally silent heterochromatin of unknown function is associated with centromeres in many organisms. In the fission yeast Schizosaccharomyces pombe, the silent mating-type loci, centromeres, and telomeres are assembled into silent heterochromatin-like domains. The Swi6 chromodomain protein affects this silencing, and now it is shown that Swi6p localizes with these three chromosomal regions. In cells lacking Swi6p, centromeres lag on the spindle during anaphase and chromosomes are lost at high rates. Thus, Swi6p is located at fission yeast centromeres and is required for their proper function.