Programmed Genome Rearrangements Research Articles

Developmentally programmed, RNA‐directed genome rearrangement in Tetrahymena

Developmentally programmed genome rearrangement has been observed in a variety of eukaryotes from vertebrates to worms to protists, and it provides an interesting exception to the general rule of the constancy of the genome. DNA elimination in the ciliated protozoan Tetrahymena is one of the most well-characterized programmed genome rearrangement events. DNA elimination in the newly formed macronucleus of Tetrahymena is epigenetically regulated by the DNA sequence of the parental macronucleus. Dicer-produced, Piwi-associated small RNAs mediate this epigenetic regulation, probably through a whole-genome comparison of the germline micronucleus to the somatic macronucleus. However, a correlation between small RNAs and programmed genome rearrangement could not be detected in the worm Ascaris suum. Therefore, different types of eukaryotes may have developed unique solutions to perform genome rearrangement.

Functional Study of Genes Essential for Autogamy and Nuclear Reorganization in Paramecium

Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.

Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling

Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.

Development and analysis of a germline BAC resource for the sea lamprey, a vertebrate that undergoes substantial chromatin diminution

Over the last several years, the sea lamprey (Petromyzon marinus) has grown substantially as a model for understanding the evolutionary fundaments and capacity of vertebrate developmental and genome biology. Recent work on the lamprey genome has resulted in a preliminary assembly of the lamprey genome and led to the realization that nearly all somatic cell lineages undergo extensive programmed rearrangements. Here we describe the development of a bacterial artificial chromosome (BAC) resource for lamprey germline DNA and use sequence information from this resource to probe the subchromosomal structure of the lamprey genome. The arrayed germline BAC library represents approximately 10x coverage of the lamprey genome. Analyses of BAC-end sequences reveal that the lamprey genome possesses a high content of repetitive sequences (relative to human), which show strong clustering at the subchromosomal level. This pattern is not unexpected given that the sea lamprey genome is dispersed across a large number of chromosomes (n approximately 99) and suggests a low-copy DNA targeting strategy for efficiently generating informative paired-BAC-end linkages from highly repetitive genomes. This library therefore represents a new and biologically informed resource for understanding the structure of the lamprey genome and the biology of programmed genome rearrangement.

Mass Culture of Paramecium tetraurelia: Figure 1.

INTRODUCTIONClonal cell lines of Paramecium tetraurelia can be established easily by single cell isolation. Given a constant supply of food, P. tetraurelia cells will remain in the vegetative phase of their life cycle and, at 27°C, will divide by binary fission every 6 h. P. tetraurelia is suitable for a range of biochemical and molecular studies such as mitochondrial genetics, post-translational tubulin modifications, variant genetic codes, programmed genome rearrangements (and their epigenetic regulation by noncoding RNAs), and RNA interference. This protocol describes the methods required to grow high-density mass cultures of P. tetraurelia in quantities sufficient to provide the material required for biochemical and molecular biological studies.

PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements inTetrahymena

Extensive DNA rearrangements occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome of Tetrahymena thermophila. To identify genes encoding proteins likely to be involved in this process, we devised a cytological screen to find proteins that specifically localize in macronuclear anlagen (Lia proteins) at the stage when rearrangements occur. We compared the localization of these with that of the chromodomain protein, Pdd1p, which is the most abundant known participant in this genome reorganization. We show that in live cells, Pdd1p exhibits dynamic localization, apparently shuttling from the parental to the developing nuclei through cytoplasmic bodies called conjusomes. Visualization of GFP-tagged Pdd1p also highlights the substantial three-dimensional nuclear reorganization in the formation of nuclear foci that occur coincident with DNA rearrangements. We found that late in macronuclear differentiation, four of the newly identified proteins are organized into nuclear foci that also contain Pdd1p. These Lia proteins are encoded by primarily novel genes expressed at the beginning of macronuclear differentiation and have properties or recognizable domains that implicate them in chromatin or nucleic acid binding. Three of the Lia proteins also localize to conjusomes, a result that further implicates this structure in the regulation of DNA rearrangement.

On the origin and functions of RNA-mediated silencing: from protists to man

Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.

RNA-Guided DNA Deletion in Tetrahymena: An RNAi-Based Mechanism for Programmed Genome Rearrangements

Ciliated protozoan are unicellular eukaryotes. Most species in this diverse group display nuclear dualism, a special feature that supports both somatic and germline nuclei in the same cell. Probably due to this unique life style, they exhibit unusual nuclear characteristics that have intrigued researchers for decades. Among them are large-scale DNA rearrangements, which restructure the somatic genome to become drastically different from its germline origin. They resemble the classical phenomenon of chromatin diminution in some nematodes discovered more than a century ago. The mechanisms of such rearrangements, their biological roles, and their evolutionary origins have been difficult to understand. Recent studies have revealed a clear link to RNA interference, and begin to shed light on these issues. Using the simple ciliate Tetrahymena as a model, this chapter summarizes the physical characterization of these processes, describes recent findings that connect them to RNA interference, and discusses the details of their mechanisms, potential roles in genome defense, and possible occurrences in other organisms.

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Abundant approximately 28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (DeltaDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These DeltaDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation DeltaDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, DeltaDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Genome Rearrangements: Mother Knows Best!

In Paramecium, developmentally programmed genome rearrangements can be altered by the presence of homologous sequences within the maternal somatic nucleus. Newly identified RNA-binding proteins appear to mediate the transfer of homologous sequence information from the maternal to the developing somatic nucleus, facilitating epigenetic regulation of this large-scale genome reorganization.

Focus on recombinational DNA repair

The Juan March workshop on ‘Recombinational DNA Repair and its Links with DNA Replication and Chromosome Maintenance’ was held in Madrid, Spain, between 13 and 15 December 2004. The workshop was organized by S. Kowalczykowski, S. West, A. Aguilera and J. Alonso. ![][1] Homologous recombination (HR) has a crucial role in chromosome maintenance by promoting error‐free repair of DNA double‐strand breaks (DSBs) and single‐strand gaps. DNA strand breaks occur spontaneously during normal cellular processes, such as DNA replication, or when cells are treated with DNA damaging agents, and are essential intermediates in several programmed genome rearrangements. HR is also required for telomere maintenance in the absence of telomerase in eukaryotes. In Escherichia coli , most of the genes involved in HR were identified in screens for recombination‐defective mutants (rec genes). Similarly, screens for radiation‐sensitive mutants in budding yeast led to the discovery of the RAD52 epistasis group genes that define the main pathway for HR in eukaryotes. The RecA and Rad51 recombinases are central to the process of HR in prokaryotes and eukaryotes, respectively. Recombinases promote homologous pairing and strand exchange, which leads to recombination between the two interacting DNA molecules (Fig 1). However, the recombinases require several other proteins to accomplish strand exchange in vivo (Kowalczykowski, 2000; Krogh & Symington, 2004; West, 2003). The role of these accessory factors in facilitating the activity of the respective recombinase was one of the topics covered at the meeting. HR and nonhomologous end joining (NHEJ) are the main pathways for repairing DSBs in eukaryotic cells, but there are distinct sub‐pathways depending on the presence of one or two ends at the break site, or flanking homologies surrounding the DSB. The regulation of these pathways of DSB repair (DSBR) is an active area of investigation, as shown by the talks at this … [1]: /embed/graphic-1.gif

Recombinational rearrangements in bacterial genome and bacterial adaptation to the environment

The rearrangement of bacterial chromosomes induced by intragenomic recombination is considered. The role of stochastic and programmed genome rearrangements in bacterial adaptation to the environment and in cell differentiation is discussed.