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

Abstract

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.

Functional Nuclear Organization of Transcription and DNA Replication: A Topographical Marriage between Chromatin Domains and the Interchromatin Compartment

“…All coding RNAs necessary for the synthesis of proteins involved in chromatin replication in the transcriptionally inert micronucleus are provided by the transcriptionally active macronucleus. Replication occurs in foci-like structures showing spatial and temporal patterns similar to the replication of ~1-Mb CDs in nuclei of higher eukaryotes (Postberg et al 2005(Postberg et al , 2008.…”

RNA-dependent control of gene amplification

“…It is difficult to imagine that differential activation of origins could occur in this structure (31). We therefore considered the possibility that copy number control, DNA reorganization, and processing could be closely linked mechanisms.…”

Radial chromatin positioning is shaped by local gene density, not by gene expression

“…Such patterns have been found evolutionarily conserved over several hundred millions of years (Alexandrova et al 2003;Federico et al 2006;Habermann et al 2001;Neusser et al 2007;Postberg et al 2005;Tanabe et al 2002) and illustrate that the radial arrangement of chromatin in the interphase nucleus represents a basic principle of nuclear architecture (for review, see Foster and Bridger 2005;Kosak and Groudine 2004;Misteli 2004;Pederson 2004;Zink 2006).…”