Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

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.     

Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences in Paramecium tetraurelia

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.     

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns.     

Invention and Mystery in Hypotrich DNA1

ABSTRACT “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas³ Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.     

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.     

Varied truncation and clustering characterize some short repeats identified in micronucleus-specific DNA of Tetrahymena thermophila

There are over 6000 internally eliminated DNA sequences (IESs) in the Tetrahymena genome that are deleted in a programmed fashion during the development of a polyploid, somatic macronucleus from a diploid germline micronucleus. Recently, based on several results, a homology and small RNA-based mechanism has been proposed for the efficient elimination of IES elements. Since the RNAi machinery is proposed to be intimately involved in silencing potentially harmful repeats such as transposons and viruses, characterization of repeats and the conditions for their developmental elimination from the somatic genome is warranted. Three short (500–600 bp) repeat families, members of which had been experimentally identified in IESs, that is, in micronucleus-specific DNA, are examined here using the Tetrahymena genome database. Members of all three families display varied degrees of truncation and are represented in macronuclear sequences. A 200 bp segment of one of the families can appear in the genome on its own, or as part of a 600 bp repeat detected experimentally, or in association with an unrelated 1 kb sequence to form a 1.2 kb repeat that is also frequently truncated. The 1 kb sequence contains a 300 bp section similar to a repeat associated with a non-long terminal repeat-like element and is often found accompanied by several more copies of this shorter repeat. These observations indicate that transposition may have had a role in the evolution of the short repeat families.     

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.     

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.     

Internal Eliminated Segments (IESs) of Oxytrichidae1

ABSTRACT Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding β-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.     

Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila

Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (DeltaLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome.     

Preferential cleavage of Paramecium DNA mediated by the C. elegans Tc1 transposase in vitro

In the ciliate Paramecium aurelia complex, thousands of internal eliminated sequences (IESs) are excised from the germline micronuclear DNA during macronuclear differentiation. Based on the resemblance of Paramecium IES end sequences to Tc1 transposon termini, it has been proposed that Paramecium IESs might have degenerately evolved from Tc1 family transposons, and still be removed by an enzyme homologous to a Tc1 transposase. In this study, we found that transposase preferentially cleaved (or nicked) 58 sites near the IESs in Paramecium DNA, at sequences consisting of TT or TCTA. Since one excision junction of the P. primaurelia W2 IES was included in such sites, this suggests that a Tc1-like transposase is involved in the IES excision process, although it is probably not a sole factor responsible for the precise cleavage. In addition, unmethylated substrate DNA appeared to decrease the cleavage specificity, suggesting an involvement of DNA methylation in the cleavage. Although these results do not directly address the transposon origin of Paramecium IESs, it is likely that the enzymatic machinery responsible for the initial cleavage is derived from a Tc1-like transposase. The mechanism necessary for precise excision is discussed, in relation to recent knowledge of IES excision obtained in Tetrahymena and Paramecium.     

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5'-TA-3' dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5'-TA-3' repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.     

Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants.     

Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia

BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.     

DNA rearrangements and mating-type determination in Paramecium tetraurelia

Ciliated protozoa have separate germline and somatic nuclei, yet unlike larger organisms, both nuclei reside in the same cytoplasm. The micronuclei contain the germline and the macronucleus is the somatic nucleus. Thousands of DNA elements are normally removed from the micronuclear genome as it forms a new macronucleus during each sexual cycle. A recent study directly links the excision of these internal eliminated sequences (IESs) to mating type determination by showing that a pleiotropic mutation affecting mating type also prevents the excision of an IES from a surface protein gene⁽¹⁾. Remarkably, once the IES is present in the old macronucleus it prevents excision of that specific IES during formation of the next macronucleus.     

A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila

Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate’s somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.     

Mutations in Pdd1 Reveal Distinct Requirements for Its Chromodomain and Chromoshadow Domain in Directing Histone Methylation and Heterochromatin Elimination

Pdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation of Tetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.