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Background: During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1., Results: These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected., Conclusions: We introduce the concept of 'RNA-induced DNA replication interference' and explain how the parental functional genome partition could become transmitted to the progeny.

Adeno-associated virus (AAV) vectors are one of the most frequently applied gene transfer systems in research and human clinical trials. Since AAV vectors do not possess an integrase activity, application is restricted to terminally differentiated tissues if transgene expression is required long term. To overcome this limitation and to generate AAV vectors that persist episomally in dividing cells, AAV vector genomes were equipped with a scaffold/matrix attachment region (S/MAR). After a mild antibiotic selection, cells transduced with AAV-S/MAR established colonies that maintained long-term transgene expression (>50 population doublings) from replicating AAV vector episomes in the absence of further selection. Unexpectedly, with a lesser but still significant efficiency, the control vector (AAV-ΔS/MAR), a standard single-stranded AAV vector, also established stable transgene-expressing colonies, most of which were maintained as replicating episomes rather than integrated vector genomes. Thus, based on the result in HeLa cells, it is concluded that AAV vector genomes per se possess the ability to establish episomal maintenance in proliferating cells, a feature that can be enhanced by incorporation of a foreign genomic element such as an S/MAR element.

Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

De novo addition of telomeric sequences can occur at broken chromosomes and must be well controlled, which is essential during programmed DNA reorganization processes. In ciliated protozoa an extreme form of DNA-reorganization is observed during macronuclear differentiation after sexual reproduction leading to the elimination of specific parts of the germline genome. Regulating these processes involves small noncoding RNAs, but in addition DNA-reordering, excision and amplification require RNA templates deriving from the parental macronucleus. We show that these putative RNA templates can carry telomeric repeats. Microinjection of RNA templates carrying modified telomeres into the developing macronucleus leads to modified telomeres in vegetative cells, providing strong evidence, that de novo addition of telomeres depends on a telomere-containing transcript from the parental macronucleus.

'If G-quadruplexes form so readily in vitro, Nature will have found a way of using them in vivo' (Statement by Aaron Klug over 30 years ago).During the last decade, four-stranded helical structures called G-quadruplex (or G4) have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological processes in vivo. The sequencing of many genomes has revealed that they are rich in sequence motifs that have the potential to form G-quadruplexes and that their location is non-random, correlating with functionally important genomic regions. In this short review, we summarize recent evidence for the in vivo presence and function of DNA and RNA G-quadruplexes in various cellular pathways including DNA replication, gene expression and telomere maintenance. We also highlight remaining open questions that will have to be addressed in the future., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting 'anchoring' non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

Background: Regulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated., Results: We show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction in Stylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression., Conclusions: Differential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.

Gene therapeutic approaches offer great opportunities to treat genetic diseases which require long-term effects after a single administration of a customized vector. For these specific approaches the optimal vector system should combine the following features: (1) it should efficiently transport the genetic cargo into target cells in vitro or in vivo, (2) it should lead to sufficient long-term expression of the therapeutic transgene, (3) it should not interfere with the expression profile or the composition of the host genome, and (4) it should not result in unwanted side effects such as immune responses or other toxic effects. Predominantly used vectors for maintenance of therapeutic DNA and long-term transgene expression in preclinical and clinical studies are based on integrase-, recombinase-, transposase- or designer nuclease-mediated somatic integration into the host genome. However, for these systems the risk of insertional mutagenesis represents a potential unwanted adverse event. Therefore, autonomously replicating genetic elements were developed and there is accumulating evidence that these episomal vectors which are maintained extrachromosomally are suitable for therapeutic applications in dividing cells. In this review we provide a state-of-the-art overview of used viral hybrid-vectors which efficiently deliver autonomous DNA (plasmid replicon pEPI and Epstein-Barr Virus-based replicons) and RNA replicons (Semliki Forest Virus replicons) into target cells. To date adenoviruses, herpesviruses and baculovirus were explored for efficient delivery of autonomous replicons into various cell types and tissues. Applications and advantages and limitations of these hybrid-vectors are discussed in this review. We believe that with further optimization autonomous replicons may play an increasingly important role in gene therapeutic applications.

The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.Molecular Therapy-Nucleic Acids (2013) 2, e118; doi:10.1038/mtna.2013.47; published online 3 September 2013.

In dividing cells, the two aims a gene therapeutic approach should accomplish are efficient nuclear delivery and retention of therapeutic DNA. For stable transgene expression, therapeutic DNA can either be maintained by somatic integration or episomal persistence of which the latter approach would diminish the risk of insertional mutagenesis. As most monosystems fail to fulfill both tasks with equal efficiency, hybrid-vector systems represent promising alternatives. Our hybrid-vector system synergizes high-capacity adenoviral vectors (HCAdV) for efficient delivery and the scaffold/matrix attachment region (S/MAR)-based pEPito plasmid replicon for episomal persistence. After proving that this plasmid replicon can be excised from adenovirus in vitro, colony forming assays were performed. We found an increased number of colonies of up to sevenfold in cells that received the functional plasmid replicon proving that the hybrid-vector system is functional. Transgene expression could be maintained for 6 weeks and the extrachromosomal plasmid replicon was rescued. To show efficacy in vivo, the adenoviral hybrid-vector system was injected into C57Bl/6 mice. We found that the plasmid replicon can be released from adenoviral DNA in murine liver resulting in long-term transgene expression. In conclusion, we demonstrate the efficacy of our novel HCAdV-pEPito hybrid-vector system in vitro and in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e83; doi:10.1038/mtna.2013.11; published online 2 April 2013.

We introduce ciliated protozoa, and more specifically the stichotrichous ciliates Oxytricha and Stylonychia, as biological model systems for the analysis of programmed DNA-reorganization processes during nuclear differentiation. These include DNA excision, DNA elimination, reordering of gene segments and specific gene amplification. We show that small nuclear RNAs specify DNA sequences to be excised or retained, but also discuss the need for a RNA template molecule derived from the parental nucleus for these processes. This RNA template guides reordering of gene segments to become functional genes and determines gene copy number in the differentiated nucleus. Since the template is derived from the parental macronucleus, gene reordering and DNA amplification are inherited in a non-Mendelian epigenetic manner.

Background: DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system., Results: In the stichotrichous ciliate Stylonychia lemnae, cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila. Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the germline micronucleus to the new macronucleus., Conclusions: We show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.

Transdifferentiation in vivo is an attractive option for autologous replacement of pancreatic β cells in patients with type 1 diabetes. It has been achieved by adenoviral delivery of genes for transcription factors in the liver and pancreas of hyperglycaemic mice. However, these viral approaches are not clinically applicable. We used the hydrodynamic approach to deliver genes Pdx1, Ngn3 (Neurog3) and MafA singly and in combination to livers of normoglycaemic rats. Five expression plasmids were evaluated. Livers were removed 1, 3, 7, 14 and 28 days after gene delivery and assayed by quantitative PCR, semi-quantitative PCR and immunohistology. Functional studies on hyperglycaemic rats were performed. The highest and most sustained expression was from a CpG-depleted plasmid (pCpG) and a plasmid with an in-frame scaffold/matrix attachment region ((pEPI(CMV)). When Pdx1, Ngn3 and MafA were delivered together to normoglycaemic rats with these plasmids, insulin mRNA was detected at all time points and was ~50-fold higher with pCpG. Insulin mRNA content of livers at days 3 and 7 was equivalent to that of a pancreas, with scattered insulin-positive cells detected by immunohistology, but levels declined thereafter. Prohormone convertase 1/3 was elevated at days 3 and 7. In hyperglycaemic rats, fasting blood glucose was lower at days 1, 3 and 7 but not thereafter, and body weight was maintained to day 28. We conclude that hydrodynamic gene delivery of multiple transcription factors to rat liver can initiate transdifferentiation to pancreatic β cells, but the process is reversible and probably requires more sustained transcription factor expression.

Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a germline micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells - many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nonviral episomal vectors represent attractive alternatives to currently used virus-based expression systems. In the late 1990s, it was shown that a plasmid containing an expression cassette linked to a scaffold/matrix attached region (S/MAR) replicates as a low copy number episome in all cell lines tested, as well as primary cells, and can be used for the genetic modification of higher animals. Once established in the cell, the S/MAR vector replicates early during S-phase and, in the absence of selection, is stably retained in the cells for an unlimited period of time. This vector can therefore be regarded as a minimal model system for studying the epigenetic regulation of replication and functional nuclear architecture. In theory, this construct represents an almost "ideal" expression system for gene therapy. In practice, S/MAR-based vectors stably modify mammalian cells with efficiencies far below those of virus-based constructs. Consequently, they have not yet found application in gene therapy trials. Furthermore, S/MAR vector systems are not trivial to handle and several critical technical issues have to be considered when modifying these vectors for various applications.

The episomal status of S/MAR (scaffold/matrix attached region)-based vectors can be confirmed by several methods including Southern blots, fluorescence in situ hybridization (FISH) analysis, or plasmid rescue experiments. In rescue experiments, genomic DNA (gDNA) or DNA from Hirt extracts is isolated from cell clones or mixed populations in which S/MAR plasmids are stably established. Bacteria are transformed with this DNA and if episomal plasmid DNA (pDNA) is present, resistant bacterial colonies will form.

As with all eukaryotic replicons, the stable establishment of S/MAR (scaffold/matrix attached region) vectors is a stochastic event that depends on poorly understood epigenetic factors such as chromatin structure and nuclear localization. Establishment efficiency describes the percentage of cells in which a particular S/MAR vector is stably retained as an episome after an initial selection period. Expected establishment efficiency for S/MAR vectors is 1-5%. This article describes a colony-forming assay that may be used either to determine establishment efficiency or to generate single cell clones.

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPβ and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Because of their high efficiency, virus-based vectors are currently used in most gene therapy trials. Because such vectors bear some potential safety risks, nonviral expression systems could be an attractive alternative. Ideally, these vectors should be completely based on chromosomal elements and replicate as an autonomous unit in the recipient cell, thus avoiding the risk of insertional mutagenesis or immunological reactions of the recipient organism. Our limited knowledge of the epigenetic regulation of replication in mammalian cells does not yet allow the rational design of such constructs. But in the late 1990s it was shown that scaffold/matrix attached region (S/MAR)-based vectors can promote episomal replication and maintenance in mammalian cells. These vectors have found broad application in basic research but are now improved for their use in the safe and reproducible genetic modification of cells and organisms and in gene therapy.

We exploit the unusual genome organization of the ciliate cell to analyze the control of specific gene amplification during a nuclear differentiation process. Ciliates contain two types of nuclei within one cell, the macronucleus and the micronucleus; and after sexual reproduction a new macronucleus is formed from a micronuclear derivative. During macronuclear differentiation, most extensive DNA reorganization, elimination, and fragmentation processes occur, resulting in a macronucleus containing short DNA molecules (nanochromosomes) representing individual genetic units and each being present in high copy number. It is believed that these processes are controlled by small nuclear RNAs but also by a template derived from the old macronucleus. We first describe the exact copy numbers of selected nanochromosomes in the macronucleus, and define the timing during nuclear differentiation at which copy number is determined. This led to the suggestion that DNA processing and copy number control may be closely related mechanisms. Degradation of an RNA template derived from the macronucleus leads to significant decrease in copy number, whereas injection of additional template molecules results in an increase in copy number and enhanced expression of the corresponding gene. These observations can be incorporated into a mechanistic model about an RNA-dependent epigenetic regulation of gene copy number during nuclear differentiation. This highlights that RNA, in addition to its well-known biological functions, can also be involved in the control of gene amplification.

An ideal vector to be used in gene therapy should allow long-term and regulated expression of the therapeutic sequence, but in many cases, it would be most desirable to remove all ectopic vector sequences from the cell once expression is no longer required. The vector pEPI is the first nonviral autonomous replicon that was constructed for mammalian cells. It represents a minimal model system to study the epigenetic regulation of replication and transcription but is also regarded as a promising alternative to currently used viral vector systems in gene therapy. Its function relies on a transcription unit linked to an S/MAR sequence. We constructed an inducible pEPI vector system based on the Tet ON system in which transcription is switched on in the presence of doxycycline. We show that for vector replication and long-term maintenance an ongoing transcription running into the S/MAR element is required. Once established, the vector is lost from the cell upon switching off transcription from the gene linked to the S/MAR. This feature provides not only controlled transgene expression but also the possibility to remove all vector molecules from the cells upon demand. This inducible episomal nonviral vector system will find broad application in gene therapy but also in reprogramming of somatic cells or modification of stem cells., (2010 Elsevier B.V. All rights reserved.)

Understanding the evolutionary origin of the nucleus and its compartmentalized architecture provides a huge but, as expected, greatly rewarding challenge in the post-genomic era. We start this chapter with a survey of current hypotheses on the evolutionary origin of the cell nucleus. Thereafter, we provide an overview of evolutionarily conserved features of chromatin organization and arrangements, as well as topographical aspects of DNA replication and transcription, followed by a brief introduction of current models of nuclear architecture. In addition to features which may possibly apply to all eukaryotes, the evolutionary plasticity of higher-order nuclear organization is reflected by cell-type- and species-specific features, by the ability of nuclear architecture to adapt to specific environmental demands, as well as by the impact of aberrant nuclear organization on senescence and human disease. We conclude this chapter with a reflection on the necessity of interdisciplinary research strategies to map epigenomes in space and time.

Background: The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins., Results: In the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes.Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts (animals, choanozoa, fungi, nuclearids, Amoebozoa). H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as Trichoplax. H3.1 and H3.1t are probably restricted to mammals.We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA) confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are conserved even in putatively early branching eukaryotic taxa (Euglenozoa/Excavata)., Conclusions: At least a basal repertoire of chromatin modifying mechanisms appears to share old common ancestry and may thus be inherent to all eukaryotes. We speculate that epigenetic principles responsive to environmental triggers may have had influenced phenotypic variation and concomitantly may potentially have had impact on eukaryotic diversification.

The discovery of autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in 1979 was considered a milestone in unraveling the regulation of replication in eukaryotic cells. However, shortly afterwards it became obvious that in Saccharomyces pombe and all other higher organisms ARSs were not sufficient to initiate independent replication. Understanding the mechanisms of replication is a major challenge in modern cell biology and is also a prerequisite to developing application-oriented autonomous replicons for gene therapeutic treatments. This review will focus on the development of non-viral episomal vectors, their use in gene therapeutic applications and our current knowledge about their epigenetic regulation.

Background: The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP) and directed into a 2000 bp long matrix attachment region sequence (MARS) derived from the human beta-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression., Results: Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element that is known to be less affected by epigenetic silencing events., Conclusions: The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

Guanine-rich sequences have been shown to readily form parallel or antiparallel G-quadruplex DNA structures in vitro. All telomeric repeat sequences contain stretches of guanine residues that can form quadruplex structures. In order to demonstrate the occurrence of the quadruplex structure in vivo, we generated by ribosome display, scFv antibodies specific for quadruplex DNA structures formed by the telomeric sequence of the ciliate Stylonychia. The macronucleus of this hypotrichous ciliate contains 10(8) telomere-capped nanochromosomes and was stained with the antibody recognizing the antiparallel G-quadruplex DNA in indirect immuno-fluorescence assays. This antibody was also used as a specific probe to study the interaction of the telomere end-binding proteins with the G-quadruplex during different stages of the cell cycle.

Background: Post-translational histone modifications (PTMs) and their specific distribution on genes play a crucial role in the control of gene expression, but the regulation of their dynamics upon gene activation and differentiation is still poorly understood. Here, we exploit the unique genome organization of ciliates to analyse PTM dynamics during gene activation in the differentiated cell and during nuclear differentiation. In the macronucleus of these cells the DNA is organized into nanochromosomes which represent independent functional units. Therefore, ciliated protozoa represent a simplistic model system to analyse the relevance of histone modifications and their localization for gene expression and differentiation., Results: We analysed the distribution of three PTMs on six individual nanochromosomes, two of which are silenced in the vegetative cell and only activated during sexual reproduction. We show that a specific relocation of these PTMs correlates with gene activation. Moreover, macronuclear-destined sequences in the differentiating macronucleus display a distribution of PTMs which differs significantly from the PTM patterns of actively transcribed genes., Conclusion: We show for the first time that a relocation of specific histone modifications takes place during activation of genes. In addition, we demonstrate that genes in a differentiating nucleus are characterised by a specific distribution and composition of PTMs. This allows us to propose a mechanistic model about the relevance of PTMs for gene activation, gene silencing and nuclear differentiation. Results described here will be relevant for eukaryotic cells in general.

Although many biochemical and structural studies have demonstrated that DNA sequences containing runs of adjacent guanines spontaneously fold into G-quadruplex DNA structures in vitro, only recently has evidence started to accumulate for their presence and function in vivo. Genome-wide analyses have revealed that functional genomic regions from highly divergent organisms are enriched in DNA sequences with G-quadruplex-forming potential, suggesting that G-quadruplexes could provide a nucleic-acid-based mechanism for regulating telomere maintenance, as well as transcription, replication and translation. Here, we review recent studies aimed at uncovering the in vivo presence and function of G-quadruplexes in genomes and RNA, with a particular focus on telomeric G-quadruplexes and how their formation and resolution is regulated to permit telomere synthesis.

Non-viral episomal vectors are regarded as attractive alternatives to currently used virus-based vectors in gene therapy. In addition, they represent a minimal model system to study the epigenetic control of basic nuclear processes, such as transcription, replication and nuclear retention. Here we analyze the dynamics of histone modifications during the cell cycle of the episomally replicating vector pEPI-eGFP. The histone code of pEPI-eGFP was compared to its integrating counterpart pGFP-C1. We found that pEPI-eGFP is preferentially associated with histone modifications typical for active chromatin, while pGFP-C1 is mostly decorated with repressive histone modifications. During interphase the distribution of histone modification on pEPI-eGFP is very non-dynamic; the S/MAR shows the highest concentration of active histone modifications. However, they are specifically removed during mitosis and this may correlate with the association and co-segregation of pEPI with the host chromosomes during cell division.

During macronuclear differentiation in ciliated protozoa, extensive DNA rearrangement and DNA excision processes occur, and these are most profound in stichotrichous ciliates, such as Stylonychia or Oxytricha. This review describes the morphological and molecular events taking place during macronuclear development in stichotrichous ciliates. Various models for the regulation of macronuclear differentiation have been proposed and will be discussed here. Finally, an attempt to speculate about the biological consequences of these rearrangement and excision processes will be made. Because specific elimination of DNA sequences not required in the differentiated nucleus can be regarded as the most extreme form of gene silencing, results obtained in these cells may also be relevant for our understanding of differentiation processes in higher eukaryotic organisms.

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained., Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization., Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis.

Since the emergence of viral resistance of hepatitis B virus (HBV) during treatment is becoming an important issue even with newer drugs, there is a need for alternative treatment options such as, for example, RNA interference (RNAi) technology. While short-term suppression of HBV replication is easily achieved with small interfering RNA oligonucleotides, this is not the case for long-term suppression due to the lack of an optimal vector system. Based on the nonviral scaffold/matrix attachment region (S/MAR)-based vector system pEPI-1, which is free of common side effects and is stably retained as an episome even in the absence of selection, we designed a short hairpin RNA (shRNA) expression vector called pEPI-RNAi for HBV suppression. HBV-replicating HepG2.2.15 cells were transfected with pEPI-RNAi, and the intracellular status of the plasmid was followed by PCR and Southern analysis. HBV replication was measured on the DNA, RNA, and protein level. HBV RNA expression was reduced by almost 85% 3 months posttransfection with pEPI-RNAi. At 8 months posttransfection in the absence of antibiotic selection pressure, the suppression level was still 70% and the vector was retained as an episome. The reduction of total intracellular HBV DNA at this point was 77%, showing a marked suppression of HBV DNA replication. At a comparable level, secretion of viral antigens, as well as progeny HBV virions, was inhibited. The S/MAR-based vector system pEPI-1 allows long-term suppression of HBV replication by the expression of suitable shRNAs. Due to its unique properties compared to commonly used vectors, it provides an interesting option for the treatment of chronically HBV-infected individuals.

Background: Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20-30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development., Methodology/principal Findings: To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways., Conclusions/significance: This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

The increasing knowledge of the molecular and genetic background of many different human diseases has led to the vision that genetic engineering might be used one day for their phenotypic correction. The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels. Manifold different vector systems for the therapeutic gene delivery have been described over the recent years. They all have their individual advantages but also their individual limitations and must be judged on a careful risk/benefit analysis. Integrating vector systems can deliver genetic material to a target cell with high efficiency enabling long-term expression of an encoded transgene. The main disadvantage of integrating vector systems, however, is their potential risk of causing insertional mutagenesis. Episomal vector systems have the potential to avoid these undesired side effects, since they behave as separate extrachromosomal elements in the nucleus of a target cell. Within this article we present a comprehensive survey of currently available episomal vector systems for the genetic modification of mammalian cells. We will discuss their advantages and disadvantages and their applications in the context of basic research, biotechnology and gene therapy.

The telomeric G-overhangs of the ciliate Stylonychia lemnae fold into a G-quadruplex DNA structure in vivo. Telomeric G-quadruplex formation requires the presence of two telomere end binding proteins, TEBPalpha and TEBPbeta, and is regulated in a cell-cycle dependent manner. Unfolding of this structure in S phase is dependent on the phosphorylation of TEBPbeta. Here we show that TEBPbeta phosphorylation is necessary but not sufficient for a G-quadruplex unfolding rate compatible with telomere synthesis. The telomerase seems to be actively involved in telomeric G-quadruplex DNA structure unfolding in vivo. Significantly, the telomerase is recruited to telomeres by phosphorylated TEBPbeta, and hence telomerase recruitment is cell-cycle regulated through phosphorylation. These observations allow us to propose a model for the regulation of G-quadruplex unfolding and telomere synthesis during the cell cycle.

It is well established that telomeres are tethered in the eukaryotic nucleus, but a detailed analysis of the regulation of telomere attachment throughout the cell cycle is still lacking. We show here that the telomeres in the macronucleus of the ciliate Stylonychia lemnae are bound to a sub-nuclear structure by an interaction of the telomere end-binding protein TEBPalpha with three SNS proteins that are integral parts of this structure. In the course of replication, the interaction of TEBPalpha with the SNS proteins is resolved and this process is regulated by cell cycle-specific phosphorylation of the SNS proteins. Our data can be incorporated into a mechanistic model for the regulation of telomere conformation and localization throughout the cell cycle.

Background: Basic functions of the eukaryotic nucleus, like transcription and replication, are regulated in a hierarchic fashion. It is assumed that epigenetic factors influence the efficiency and precision of these processes. In order to uncouple local and long-range epigenetic features we used an extra-chromosomal replicon to study the requirements for replication and segregation and compared its behavior to that of its integrated counterpart., Results: The autonomous replicon replicates in all eukaryotic cells and is stably maintained in the absence of selection but, as other extra-chromosomal replicons, its establishment is very inefficient. We now show that following establishment the vector is stably associated with nuclear compartments involved in gene expression and chromosomal domains that replicate at the onset of S-phase. While the vector stays autonomous, its association with these compartments ensures the efficiency of replication and mitotic segregation in proliferating cells., Conclusion: Using this novel minimal model system we demonstrate that relevant functions of the eukaryotic nucleus are strongly influenced by higher nuclear architecture. Furthermore our findings have relevance for the rational design of episomal vectors to be used for genetic modification of cells: in order to improve such constructs with respect to efficiency elements have to be identified which ensure that such constructs reach regions of the nucleus favorable for replication and transcription.

During macronuclear differentiation in ciliated protozoa, most amazing "DNA gymnastics" takes place, which includes DNA excision, DNA elimination, DNA reorganization, and DNA-specific amplification. Although the morphological events occurring during macronuclear development are well described, a detailed knowledge of the molecular mechanisms and the regulation of this differentiation process is still missing. However, recently several models have been proposed for the molecular regulation of macronuclear differentiation, but these models have yet to be verified experimentally. The scope of this review is to summarize recent discoveries in different ciliate species and to compare and discuss the different models proposed. Results obtained in these studies are not only relevant for our understanding of nuclear differentiation in ciliates, but also for cellular differentiation in eukaryotic organisms in general as well as for other disciplines such as bioinformatics and computational biology.

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Although the genetic therapy of human diseases has been conceptually possible for many years we still lack a vector system that allows safe and reproducible genetic modification of eukaryotic cells and ensures faithful long-term expression of transgenes. There is increasing agreement that vectors that are based exclusively on chromosomal elements, which replicate autonomously in human cells, could fulfill these criteria. The rational construction of such vectors is still hindered by our limited knowledge of the factors that regulate chromatin function in eukaryotic cells. This review sets out to summarize how our current knowledge of nuclear organization can be applied to the design of extrachromosomal gene expression vectors that can be used for human gene therapy. Within the past years a number of episomal nonviral constructs have been designed and their replication strategies, expression of transgenes, mitotic stability, and delivery strategies and the mechanisms required for their stable establishment will be discussed. To date, these nonviral vectors have not been used in clinical trials. Even so, many compelling arguments can be developed to support the view that nonviral vector systems will play a major role in future gene therapy protocols.

Ciliates have a somatic and a germline nucleus; after sexual conjugation a new somatic nucleus forms from the new zygotic germline nucleus. Formation of the somatic nucleus involves precise elimination of a large portion of DNA sequences from the germline. Here we compare the architecture of the germline and somatic versions of the actin I gene in two geographically isolated strains of Stylonychia lemnae. We show that the structure of the germline gene is surprisingly mercurial, with the distinction between germline-limited and somatic sequences variable over the course of evolution. This is, to our knowledge, the first example of evolutionary swapping of retained versus deleted sequences during ciliate development, with sequences deleted during development that are specifically retained in another strain.

In contrast to the chromosomal genome organization common to most eukaryotes, DNA in the macronucleus of spirotrichous ciliates like Stylonychia lemnae is organized into small gene-sized nanochromosomes. We intended to elucidate whether a spatial organization of nucleoli similar to other eukaryotes can be found in absence of typical chromosomes. Whereas micronuclei of Stylonychia exhibit homogenously stained heterochromatin and possess no nucleoli, macronuclear chromatin is compartmentalized and contains numerous putative nucleoli. Since the identity of these spherical structures has never been unequivocally demonstrated to date, we applied immunofluorescence techniques together with confocal laser scanning microscopy to identify nucleolar bodies in the macronucleus of Stylonychia and to analyse their spatial organization. We found that multiple spherical bodies, which fulfil nucleolar function, occupy a peripheral localization in mature macronuclei. Using fibrillarin/Nop1p as a nucleolar marker, we monitored the assembly of such nucleolar bodies during macronuclear differentiation. 3D-FISH experiments revealed that rRNA genes are mostly concentrated adjacent to but not inside of fibrillarin/Nop1p-containing bodies. We further showed that transcription sites for rRNA synthesis but also for mRNA synthesis occur predominantly at surfaces of nucleolar bodies and chromatin-poor spaces bordering condensed chromatin. Our data suggest that transcription of rRNA genes in the macronucleus of Stylonychia does not rely on a classical nucleolus-type organization. We assume that vectorial synthesis and processing of rRNA and mRNA is directed to a functional interchromatin compartment.

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

After sexual reproduction in ciliated protozoa a new macronucleus differentiates from a micronuclear derivative. In the course of macronuclear development dramatic DNA- and chromatin reorganisation processes occur, which include splicing of DNA sequences such as IES (internal eliminated sequences) and transposon-like elements during formation of polytene chromosomes, degradation of the polytene chromosomes and specific elimination of micronuclear-specific DNA, de novo addition of telomeres and specific amplification of DNA sequences. In order to understand the molecular basis of this nuclear differentiation process, analysis of developmentally regulated genes seems to be a necessary prerequisite. We performed a microarray analysis to identify genes differentially expressed during macronuclear differentiation. 467 sequences from cDNA libraries were identified as possible candidates from which 384 sequences were further characterised by sequence analysis. These sequences were identified, if possible, by DNA and protein BLAST analysis. Expression of one of these sequences was silenced by RNAi and a preliminary functional analysis performed. Results presented in this study provide the basis for a functional characterisation of genes differentially expressed during this nuclear differentiation process.

Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPalpha and TEBPbeta. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPalpha in attaching telomeres in the nucleus and in recruiting TEBPbeta to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPbeta in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle-dependent phosphorylation of TEBPbeta.

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.