Exploiting nuclear duality of ciliates to analyse topological requirements for DNA replication and transcription.

Spatial and temporal replication patterns are used to describe higher-order chromatin organisation from nuclei of early metazoan to mammalian cells. Here we demonstrate evolutionary conserved similarities and differences in replication patterns of micronuclei and macronuclei in the spirotrichous ciliate Stylonychia lemnae. Since this organism possesses two kinds of morphologically and functionally different nuclei in one cell, it provides an excellent model system to analyse topological requirements for DNA replication and transcription. Replication in the heterochromatic micronucleus occurs in foci-like structures showing spatial and temporal patterns similar to nuclei of higher eukaryotes, demonstrating that these patterns are inherent features of nuclear architecture. The 'nanochromosomes' of the macronucleus are replicated in the propagating replication band. We show that it consists of hundreds of replication foci. Post-replicative macronuclear chromatin remains organised in foci. These foci are not randomly distributed throughout the macronucleus, indicating a higher-order organisation of macronuclear chromatin above the level of 'nanochromosomes'. Both telomerase and proliferating cell nuclear antigen (PCNA) occur as foci-like structures in the rear zone of the replication band, suggesting that a wave of chromatin modification driven by a short or continuous exogenous signal permits the assembly of replication factories at predicted sites. We further show that transcription occurs at discrete sites colocalised with putative nucleoli and dispersed chromatin. Common principles of functional nuclear architecture were conserved during eukaryotic evolution. Moreover nuclear duality inherent to ciliates with their germline micronucleus and their somatic macronucleus may eventually provide further insight into epigenetic regulation of transcription, replication and nuclear differentiation.