The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

SummaryThe mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3Sp, the ortholog of budding yeast Scm3Sc. Scm3Sp depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3Sp coaffinity purifies with CENP-ACnp1 and associates with CENP-ACnp1 in vitro, yet localizes independently of intact CENP-ACnp1 chromatin and is differentially released from chromatin. While Scm3Sc has been proposed to form a unique hexameric nucleosome with CENP-ACse4 and histone H4 at budding yeast point centromeres, we favor a model in which Scm3Sp acts as a CENP-ACnp1 receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-ACnp1 from the Sim3 escort and mediate assembly of CENP-ACnp1 into subkinetochore chromatin.

Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A Cnp1 and kinetochore assembly over the central domain. The H3K9methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A Cnp1 chromatin on naïve templates. Once assembled, CENP-A Cnp1 chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified.Metazoan centromeres are mostly composed of repetitive DNA upon which the kinetochore assembles to mediate chromosome segregation. Epigenetic factors contribute to the establishment and maintenance of kinetochores at particular sites, which are composed of CENP-A chromatin (1-4). However, the primary signals specifying the site of CENP-A chromatin, and thus kinetochore assembly, are unknown.In Drosophila, human, and fission yeast, kinetochores are embedded in heterochromatin (5-7). Fission yeast centromeres have two distinct domains: outer repeats (otr) that flank the central kinetochore domain composed of innermost repeats (imr) and central core (cnt/cc) DNA (3, 8) (Fig. 1A).Heterochromatin, containing Swi6 (heterochromatin protein 1 or HP1) bound to histone H3 dimethylated on lysine 9 (H3K9me2), forms over the otr, whereas CENP-A Cnp1 replaces histone H3 in the central domain (5, 7, 9-11). Centromeric heterochromatin contributes to centromere function by recruiting cohesin and mediating cohesion between sister centromeres (12, 13). Here, we test the idea that heterochromatin marks sites for CENP-A chromatin assembly (14) Fission yeast minichromosomes must contain at least part of an otr and a large portion of central domain (imr-cc) DNA to allow kinetochore assembly, segregation function, and minichromosome retention (3,(14)(15)(16)(17). Small interfering RNAs (siRNAs) originating from noncoding otr transcripts direct H3K9 methylation and heterochromatin formation on these repeats (8, 18). Defects in various components cause loss or reduction in H3K9 methylation, Swi6, and centromeric cohesin (5, 9, 13). Endogenous chromosomes segregate without centromeric heterochromatin, because sister chromatids remain tethered by arm cohesin. However, mitotic stability of small circular minichromosomes is obliterated (19). Minichromosomes containing otr and cc DNA have centromere activity, resulting in a low lo...