The Organisation of Fission Yeast Centromeres

Large blocks of heterochromatin are prevalent at the centromere regions of many eukaryotes. In metazoa, large arrays of repetitive DNA of up to several megabase pairs are packaged as heterochromatin at centromeres. The structure of S. pombe centromeres is somewhat similar to that of more complex eukaryotes in that they are also relatively large, repetitive and complex structures which occupy 35-110 kb (Steiner et al. 1993; Takahashi et al. 1992). This is in contrast to the comparatively simple point centromeres of the budding yeast Saccharomyces cerevisiae which are only 125 bp (Cleveland et al. 2003; Sullivan et al. 2001). Fission yeast kinetochores bind 2-4 microtubules at mitosis (Ding et al. 1993). This is again more reminiscent of the multiple microtubule interactions to each kinetochore in metazoa than the single microtubule attachment observed in budding yeast (Winey et al. 1995). S. pombe centromeres are composed of a unique central core (cc) of 4-7 kb which is flanked by the innermost repeats (imrL/R) and the outer repeats on which centromeric het-erochromatin forms (Steiner et al. 1993; Takahashi et al. 1992; Allshire et al. 1995; Partridge et al. 2000; Fig. 1). Together the central core and imr repeats make up the central domain and are packaged in a centromere-specific form of chromatin containing the histone H3 variant Cnp1 (the CENP-A homologue in fission yeast), which replaces histone H3 (Takahashi et al. 2000). This central domain has an unusual chromatin structure as partial digestion with micrococcal nuclease produces a smeared pattern rather than the typical ladder pattern (Polizzi and Clarke 1991; Takahashi et al. 1992). Genes are also silenced when placed in this central domain, but the factors involved are distinct from those that affect heterochromatin formation on the outer repeats (Allshire et al. 1994, 1995; Ekwall et al. 1996; Partridge et al. 2000; Pidoux et al. 2003). Thus, this central domain is functionally and structurally distinct from the heterochromatic outer repeat regions (Allshire et al. 1995; Partridge et al. 2000). The central core itself is essential for centromere activity, but alone it is not sufficient to assemble an active centromere. Studies using minichromosomes have demonstrated that at least part of the heterochromatic outer repeat, in combination with central domain sequences, is essential to allow the de novo formation of active centromeres (Baum et al. 1994; Ngan and Clarke 1997; Takahashi et al. 1992).

Fig. 1 Centromere organisation in fission yeast. The central core (cc) is flanked by inverted innermost repeats (imr) the sequence of which is unique to each centromere. Together cc and imr form the central domain, which is the site of kinetochore formation, and in this region most histone H3 is replaced by the H3 variant Cnpl (mammalian CENP-A). The central domain is flanked by arrays of inverted repeats, the number and organisation of which vary at each centromere although the sequence is similar. These outer repeat regions (dg/dh) are packaged as heterochromatin in which lysine residues in the N-terminal tails of histones H3 and H4 are hypoacetylated. H3 is dimethyl-ated on lysine 9 (H3K9me2) by the histone methyltransferase Clr4. This H3K9me2 recruits the chromo domain protein Swi6, which causes heterochromatin to spread and is required for the association of the cohesin complex. Both the RITS and RDRC complexes are known to associate with the centromeric outer repeats. Dark blue vertical lines denote the position of tRNA gene clusters

Fig. 1 Centromere organisation in fission yeast. The central core (cc) is flanked by inverted innermost repeats (imr) the sequence of which is unique to each centromere. Together cc and imr form the central domain, which is the site of kinetochore formation, and in this region most histone H3 is replaced by the H3 variant Cnpl (mammalian CENP-A). The central domain is flanked by arrays of inverted repeats, the number and organisation of which vary at each centromere although the sequence is similar. These outer repeat regions (dg/dh) are packaged as heterochromatin in which lysine residues in the N-terminal tails of histones H3 and H4 are hypoacetylated. H3 is dimethyl-ated on lysine 9 (H3K9me2) by the histone methyltransferase Clr4. This H3K9me2 recruits the chromo domain protein Swi6, which causes heterochromatin to spread and is required for the association of the cohesin complex. Both the RITS and RDRC complexes are known to associate with the centromeric outer repeats. Dark blue vertical lines denote the position of tRNA gene clusters

The outer repeats (otr) themselves are composed of two elements, known as the dh and dg (or K and L) repeats, which are arranged differently with respect to each other at each centromere (Steiner et al. 1993; Takahashi et al. 1992). Because these repeats are packaged into heterochromatin, expression levels of marker genes (ade6+ and ura4+, for example) inserted at sites across the outer repeats are subject to variable repression or expression, resulting in phenotypic variegation, and this has allowed the development of screens to identify many factors involved in heterochromatin and hence centromere structure and function (Allshire et al. 1995; Ekwall et al. 1999).

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