Nuclear spatial organization influences centromere identity

Abstract

Centromeres are specialized chromosomal domains that provide the attachment site for spindle microtubules emanating from opposite spindle pole bodies (SPBs; centrosome equivalent). Centromere identity is epigenetically defined by the histone H3-variant, CENP-A. Both genetic and epigenetic factors influence the de novo assembly of CENP-A. In the fission yeast, Schizosaccharomyces pombe, CENP-ACnp1 (Cnp1, ortholog of human CENP-A) chromatin prefers to establish on centromere central domain DNA, but it also can assemble on non-centromeric DNA in rare situations. Adjacent heterochromatin, formed over flanking outer repeats, is required to establish CENP-ACnp1 chromatin on central domain, but overexpression of CENP-ACnp1 can bypass this requirement. All constitutive heterochromatic loci (centromeres, telomeres and mating-type regions and synthetic heterochromatin) preferentially localize to the nuclear envelope (NE) or SPB. Newly synthesized CENP-ACnp1 replaces S-phase deposited placeholder H3 when it is incorporated during the subsequent G2 phase into centromeric central domain chromatin which is transcribed by RNAPII (RNA polymerase II). These observations suggest that heterochromatin might influence CENP-ACnp1 chromatin establishment by modifying central domain chromatin properties or exposing it to favorable nuclear locations for CENP-ACnp1 assembly. All three centromeres are clustered at the SPBs and centromere-associated CENP-ACnp1 assembly factors including HJURPScm3, RbAP46/48Mis16, Mis18, Eic1/Mis19 are all concentrated at this location in G2 when new CENP-ACnp1 is incorporated. The aim of my thesis is to investigate the influence of nuclear positioning of heterochromatin on CENP-ACnp1 establishment in fission yeast. I show that minichromosomes carrying heterochromatic outer repeats are located close to SPBs during interphase, consistent with this location influencing CENP-ACnp1 incorporation. CENP-ACnp1 is established on central domain inserted in cis close to, but not far away from, functional endogenous centromeres or neocentromeres. Neocentromeres also join the centromere cluster at SPBs during interphase. CENP-ACnp1 establishment is not dependent on the local chromatin context or DNA sequence at insertion sites. Moreover, direct tethering of central domain-bearing plasmids in trans to SPBs promotes de novo CENP-ACnp1 and kinetochore proteins assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation. Heterochromatin is not required for the establishment of CENP-ACnp1 on central domain placed in cis or trans close to the SPB-centromere clusters. Collectively, I conclude that heterochromatin mediates the association of adjacent central domain with SPBs thus exposing it to high concentrations of CENP-ACnp1 and associated assembly factors to promote de novo CENP-ACnp1 chromatin and kinetochores assembly. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.