Franziska Jönsson

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI‐1, a vector in which a chromosomal scaffold/matrix‐attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T‐antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI‐1 by crosslinking with cis‐diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south‐western analysis, the vector shows exclusive binding to hnRNP‐U/SAF‐A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA–protein complex demonstrates that pEPI‐1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.

RNA-dependent control of gene amplification

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.

The Pathway to Detangle a Scrambled Gene

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 Draft Assembly of the Radically Organized Stylonychia lemnae Macronuclear Genome

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 use of RNAI to analyze gene function in spirotrichous ciliates

The use of small double-stranded RNA to inhibit expression of genes has proven to be a powerful tool to analyze the biological function of genes in various eukaryotic organisms. We have established the RNAi technology in three genera of spirotrichous ciliates, Euplotes, Oxytricha and Stylonychia. Here we describe this method to silence gene expression in these three genera. Expression of the following genes was inhibited: the catalytic subunit of telomerase (TERT) and the telomerase-associated protein p43 in Euplotes and the a-subunit of the telomere-binding protein (αTeBP) of Stylonychia and Oxytricha. In addition, we report how the biological function of a developmentally regulated gene can be elucidated by this technique in Stylonychia.

The telomerase-associated protein p43 is involved in anchoring telomerase in the nucleus

Telomere replication of eukaryotic chromosomes is achieved by a specialized enzyme, the telomerase. Although the biochemistry of end-replication is well understood, little is known about the organization of the end-replication machinery, its regulation throughout the cell cycle or the biological function of the telomerase-associated proteins. Here we investigate the function of the telomerase-associated protein p43 within the macronucleus of the ciliated protozoa Euplotes. It has been shown that p43 binds in vitro to the RNA subunit of telomerase and shares homology with the La autoantigen family. It therefore has been suggested that it is involved in the assembly and/or nuclear retention of telomerase. We show that the p43-telomerase complex is bound to a subnuclear structure in vivo and is resistant to electroelution. Upon inhibition of p43 or telomerase expression by RNAi, which in this study was used for the first time in spirotrichs, this complex is no longer retained in the nucleus. Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.

The Unusual Way to Make a Genetically Active Nucleus

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.

Interconversion of Germline-Limited and Somatic DNA in a Scrambled Gene

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.

Organization of the macronuclear gene-sized pieces of stichotrichous ciliates into a higher order structure via telomere–matrix interactions

Macronuclear DNA of stichotrichous ciliates occurs in small ‘gene-sized’ molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 108 DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere–matrix interactions in higher eukaryotes.

RNA-template dependent de novo telomere addition.

Abstract 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.