Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1-7. How local chromatin interactions govern higher-order folding of chromatin fibers and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form “globules”. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9-11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fiber compaction at centromeres and promotes prominent interarm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

Summary The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either CENP-A’s amino- or carboxy-terminal tails for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

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...