LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

FANCJ promotes DNA synthesis through G‐quadruplex structures

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ^−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.     

Role of histone deacetylation in developmentally programmed DNA rearrangements in Tetrahymena thermophila

In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.     

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.     

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.     

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.     

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.     

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes,a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.