Domesticated piggyBac Transposase Research Articles

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

Coupling DNA Damage and Repair: an Essential Safeguard during Programmed DNA Double-Strand Breaks?

DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the 'price to pay' to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.

Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium.

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.

A germline-limited piggyBac transposase gene is required for precise excision in Tetrahymena genome rearrangement.

Developmentally programmed genome rearrangement accompanies differentiation of the silent germline micronucleus into the transcriptionally active somatic macronucleus in the ciliated protozoan Tetrahymena thermophila. Internal eliminated sequences (IES) are excised, followed by rejoining of MAC-destined sequences, while fragmentation occurs at conserved chromosome breakage sequences, generating macronuclear chromosomes. Some macronuclear chromosomes, referred to as non-maintained chromosomes (NMC), are lost soon after differentiation. Large NMC contain genes implicated in development-specific roles. One such gene encodes the domesticated piggyBac transposase TPB6, required for heterochromatin-dependent precise excision of IES residing within exons of functionally important genes. These conserved exonic IES determine alternative transcription products in the developing macronucleus; some even contain free-standing genes. Examples of precise loss of some exonic IES in the micronucleus and retention of others in the macronucleus of related species suggest an evolutionary analogy to introns. Our results reveal that germline-limited sequences can encode genes with specific expression patterns and development-related functions, which may be a recurring theme in eukaryotic organisms experiencing programmed genome rearrangement during germline to soma differentiation.

Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements.

During sexual processes, the ciliate Paramecium eliminates 25–30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.

Programmed Genome Rearrangements in Tetrahymena.

Ciliates are champions in programmed genome rearrangements. They carry out extensive restructuring during differentiation to drastically alter the complexity, relative copy number, and arrangement of sequences in the somatic genome. This chapter focuses on the model ciliate Tetrahymena, perhaps the simplest and best-understood ciliate studied. It summarizes past studies on various genome rearrangement processes and describes in detail the remarkable progress made in the past decade on the understanding of DNA deletion and other processes. The process occurs at thousands of specific sites to remove defined DNA segments that comprise roughly one-third of the genome including all transposons. Interestingly, this DNA rearranging process is a special form of RNA interference. It involves the production of double-stranded RNA and small RNA that guides the formation of heterochromatin. A domesticated piggyBac transposase is believed to cut off the marked chromatin, and the retained sequences are joined together through nonhomologous end-joining processes. Many of the proteins and DNA players involved have been analyzed and are described. This link provides possible explanations for the evolution, mechanism, and functional roles of the process. The article also discusses the interactions between parental and progeny somatic nuclei that affect the selection of sequences for deletion, and how the specific deletion boundaries are determined after heterochromatin marking.

The taming of the shrew: Regulation of a catalytically active domesticated transposase.

Transposons are mobile genetic elements that can be harmful for the host when mobilized. However, they are also genomic reservoirs for novel genes that can be evolutionarily beneficial. There are many examples of domesticated transposases, which play important roles in the hosts. In most cases domesticated transposases have lost their endonuclease activities and the hosts utilize their DNA-binding properties. However, some other domesticated transposases perform endonuclease activities for host biological processes. Because such a catalytically active transposase is potentially harmful for the integrity of the host genome, its activity should be tightly regulated. The catalytically active domesticated piggyBac transposase Tpb2p catalyzes programmed DNA elimination in the ciliate Tetrahymena. Here, we discuss the regulatory mechanism that prevents unintended DNA cleavage by Tpb2p and compare it to another well-studied catalytically active domesticated transposase, the RAG recombinase in V(D)J recombination. The regulatory mechanisms involve the temporarily regulated expression of the transposases, the target sequence preference of the endonuclease, and the recruitment of the transposases to locally restricted chromatin environments.

A Domesticated PiggyBac Transposase Interacts with Heterochromatin and Catalyzes Reproducible DNA Elimination in Tetrahymena

The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p.

The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway.

Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes.

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.

Subtraction by addition: domesticated transposases in programmed DNA elimination: Figure 1.

The ciliate Paramecium tetraurelia must eliminate approximately 60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478-2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed silencing machinery can guide major genome reorganization.