Both central core and outer repeat regions are required for de novo formation of a functional centromere when naked DNA is transformed into fission yeast. On the other hand, genes encoding central core proteins such as mis6, mis12, mal2, cnp1 and sim4 are essential, yet virtually all outer repeat proteins are non-essential (for instance swi6, clr4, chp1, rik1, tas3, ago1, dcr1 and rdp1 RNAi components). What, then, is the function of the twin outer repeat domains?
One known function of the outer repeats is in cohesion. swi6
mutants prematurely lose cohesion at the centromere, but show no defect in cohesion along the chromosome arms. Consistent with this, chromatin immunoprecipitation indicates that in a swi6 mutant, Rad21-Cohesin is lost specifically from the heterochromatic outer repeats (and other heterochromatin sites) but other sites are unaffected (Bernard et al. 2001
; Nonaka et al. 2002
mutants are synthetically lethal. In a rad21
mutant at the permissive temperature, there is enough residual cohesin function to ensure segregation function, though there is a high incidence of lagging chromosomes. In the absence of Swi6, centromeric cohesion is lost, but arm cohesion is adequate to ensure a sufficient level of chromosome bi-orientation. In the absence of both Swi6 and Rad21, the lack of centromeric cohesion and the reduced arm cohesion are so debilitating that the accurate chromosome slips below a level that is compatible with viability. Swi6 has been shown to interact physically with another subunit of cohesin, Psc3 (Nonaka et al. 2002
). Thus, Swi6-containing heterochromatin is responsible for recruiting a high density of cohesin to the centromeric outer repeats. Interestingly, this link seems to be conserved since vertebrate cells lacking dicer display disrupted heterochromatin and defective centromeric cohesion (Fukagawa et al. 2004
Fission yeast centromere 1 is almost perfectly symmetrical (Wood et al. 2002
): the imr
sequences are perfect inverted repeats (98% identical), as are the outer repeats; nucleotide changes on one side are often mirrored on the other. The imr
sequences of centromeres 2 and 3 are also perfect inverted repeats, though there are different numbers of otr
repeats on the left and right sides of the two centromeres. The symmetry has prompted speculation that the two sides of the centromere interact in some way to form a higher order structure such as a loop (Fishel et al. 1988
; Takahashi et al. 1992
; Clarke et al. 1993
). Might heterochromatin and the cohesin it recruits have a role in the architecture of such a loop? Possibly it has a role in holding together not only sister chromatids, but in intramolecular synapsis of the left and right sides of the centromere. What would the purpose of such a loop be? Perhaps this architecture would ensure that the central core is presented optimally for kinetochore assembly (). Mutants in RNAi, heterochromatin and cohesin components have a high incidence of lagging chromosomes (chromatids) on late anaphase spindles (Ekwall et al. 1995
; Hall et al. 2003
; Volpe et al. 2003
). In larger eukaryotes, lagging chromosomes have been shown to be merotelically oriented—this is when at single kinetochore is simultaneously attached to MTs emanating from both spindle poles (reviewed in Pidoux & Allshire 2003
). Kinetochore stretching occurs, and there is failure or delay in segregating to the poles. Merotely is a major contributor to aneuploidy in mammalian tissue culture cells (Cimini et al. 2001
). Fission yeast kinetochores are each associated with two to four MTs (Ding et al. 1993
) and the behaviour of lagging chromosomes observed in living cells is consistent with merotelic attachment (Pidoux et al. 2000
). The hypothetical loop structure may be important for the imposition of rigidity on the kinetochore, ensuring that the multiple MT binding sites on each kinetochore are clamped together so that they all face the same direction and thus promote monopolar (amphitelic) attachment. Another protein which may influence centromere orientation is Pcs1 (Rabitsch et al. 2003
). Its homologue in budding yeast, Csm1, is part of the monopolin complex required for mono-orientation of sister kinetochores in meiosis I, and is thought to act in clamping the MT-attachment sites together. Pcs1 does not seem to be required in fission yeast meiosis I, but its absence in mitosis causes lagging chromosomes, suggesting that it could play a role in clamping adjacent MT attachment sites together on a kinetochore.
Figure 5 Speculative models for the role of outer repeat heterochromatin in centromere specification and architecture. (a) The de novo assembly of kinetochores might be facilitated by heterochromatic ‘signposts’ which instruct the cell to incorporate (more ...)
As we have seen, cells with compromized outer repeat heterochromatin have chromosome segregation defects but they are viable. Yet when plasmids are transformed into cells, both central core and outer repeat elements are essential for segregation function. Perhaps the outer repeats are required initially in the establishment of a functional centromere to tell the cell where to deposit CENP-A for the assembly of the kinetochore (). Then the essential function of the outer repeats may be over, and its roles may be limited to those important but not absolutely essential roles described above—cohesion (arm cohesion suffices) and prevention of merotely (lagging chromosomes occur, but accurate segregation is at a level compatible with viability). Obviously, in non-experimental situations, the cell is not going to encounter naked DNA upon which to assemble a kinetochore. Perhaps the outer repeats function in a kind of ‘reset’ mechanism that can boost centromere architecture if the structure becomes weakened, or there is reduced CENP-A for some reason.
These ideas are testable now that the ability to make ‘synthetic’ heterochromatin exists (Schramke & Allshire 2003
). Can a centromere form when a central core is placed between two blocks of synthetic heterochromatin, for instance? Once a centromere is assembled, can outer repeat sequences be removed, or the trigger for synthetic heterochromatin switched off? The pioneering experiments of Mitsuhiro Yanagida and Louise Clarke should be revisited in the light of our knowledge about protein domains of the centromere, the nature and genesis of heterochromatin and the functional significance of CENP-A, and the specialized central core chromatin.
The kinetochore is formed over the central core chromatin, and this is where MTs would be expected to contact the centromere; indeed the microtubule-associated protein (MAP) Dis1 has been localized to this region by ChIP. However, another MAP, Alp14, is located at the imr
regions. While both proteins associate with the centromere in a mitosis dependent fashion, only Alp14's association is fully dependent on MTs (Garcia et al. 2001
; Nakaseko et al. 2001
). Perhaps these differences are a reflection of multiple types of MT interaction with the kinetochore.
and histone H3 are interspersed on experimentally produced extended chromatin fibres, yet in fixed cells they appear in cytologically distinct domains as juxtaposed cylinders or layers (Blower et al. 2002
). The difference between the two observations is explicable by a model in which the centromeric chromatin loops or coils so that all the CENP-A domains are in register, forming a surface for kinetochore assembly and MT interaction. If the fission yeast centromere does indeed form a loop structure as we propose, it might represent a single kintochore unit, interacting with two to four mts, whereas higher eukaryotic centromeres, which are known to be modular structures (Zinkowski et al. 1991
; Blower et al. 2002
), would consist of re-iterated units.