Genome organization: Raison d'être of ancestral linkage groups.

The genomes of extant organisms contain conserved blocks of regions that can be traced back to ancient ancestors, yet the evolutionary pressures that maintained such genomic segments remain unclear. New research on a curious organism with two different genomes sheds light on why our genomes are organized as they are.

A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination.

Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena, the interaction between Piwi-associated small RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.

The genome-wide meiotic recombination landscape in ciliates and its implications for crossover regulation and genome evolution.

Meiotic recombination is essential for sexual reproduction and its regulation has been extensively studied in many taxa. However, genome-wide recombination landscape has not been reported in ciliates and it remains unknown how it is affected by the unique features of ciliates: the synaptonemal complex (SC)-independent meiosis and the nuclear dimorphism. Here, we show the recombination landscape in the model ciliate Tetrahymena thermophila by analyzing single-nucleotide polymorphism datasets from 38 hybrid progeny. We detect 1021 crossover (CO) events (35.8 per meiosis), corresponding to an overall CO rate of 9.9 cM/Mb. However, gene conversion by non-crossover is rare (1.03 per meiosis) and not biased towards G or C alleles. Consistent with the reported roles of SC in CO interference, we find no obvious sign of CO interference. CO tends to occur within germ-soma common genomic regions and many of the 44 identified CO hotspots localize at the centromeric or subtelomeric regions. Gene ontology analyses show that CO hotspots are strongly associated with genes responding to environmental changes. We discuss these results with respect to how nuclear dimorphism has potentially driven the formation of the observed recombination landscape to facilitate environmental adaptation and the sharing of machinery among meiotic and somatic recombination.

A family of carboxypeptidases catalyzing α- and ß-tubulin tail processing and deglutamylation.

Tubulin posttranslational modifications represent an important mechanism involved in the regulation of microtubule functions. The most widespread among them are detyrosination, α∆2-tubulin, and polyglutamylation. Here, we describe a family of tubulin-modifying enzymes composed of two closely related proteins, KIAA0895L and KIAA0895, which have tubulin metallocarboxypeptidase activity and thus were termed TMCP1 and TMCP2, respectively. We show that TMCP1 (also known as MATCAP) acts as α-tubulin detyrosinase that also catalyzes α∆2-tubulin. In contrast, TMCP2 preferentially modifies ßI-tubulin by removing three amino acids from its C terminus, generating previously unknown ßI∆3 modification. We show that ßI∆3-tubulin is mostly found on centrioles and mitotic spindles and in cilia. Moreover, we demonstrate that TMCPs also remove posttranslational polyglutamylation and thus act as tubulin deglutamylases. Together, our study describes the identification and comprehensive biochemical analysis of a previously unknown type of tubulin-modifying enzymes involved in the processing of α- and ß-tubulin C-terminal tails and deglutamylation.

Programmed DNA elimination: New metazoan models.

Programmed DNA elimination (PDE) occurs in various metazoans. Parasitic nematodes have long been the major experimental model for PDE investigation. New studies have reported that some genetically tractable free-living nematodes also undergo PDE, paving the way for understanding the molecular mechanisms of PDE in metazoans.

PIWI-Directed DNA Elimination for Tetrahymena Genetics.

Piwi-bound small RNAs induce programmed DNA elimination in the ciliated protozoan Tetrahymena. Using the phenomenon called codeletion, this process can be reprogrammed to induce ectopic DNA elimination at basically any given genomic location. Here, we describe the usage of codeletion for genetic studies in Tetrahymena and for investigations of the molecular mechanism of Piwi-directed programmed DNA elimination.

Absolute quantification of chromosome copy numbers in the polyploid macronucleus of Tetrahymena thermophila at the single-cell level.

Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by amitosis, making ciliates a potentially valuable model system to study this process. However, a method to accurately quantify the copy number of MAC chromosomes has not yet been established. Here, we used droplet digital PCR (ddPCR) to quantify the absolute copy number of the MAC chromosomes in Tetrahymena thermophila. We first confirmed that ddPCR is a sensitive and reproducible method to determine accurate chromosome copy numbers at the single-cell level. We then used ddPCR to determine the copy number of different MAC chromosomes by analyzing individual T. thermophila cells in the G1 and the amitotic (AM) phases. The average copy number of MAC chromosomes was 90.9 at G1 phase, approximately half the number at AM phase (189.8). The copy number of each MAC chromosome varied among individual cells in G1 phase and correlated with cell size, suggesting that amitosis accompanied by unequal cytokinesis causes copy number variability. Furthermore, the fact that MAC chromosome copy number is less variable among AM-phase cells suggests that the copy number is standardized by regulating DNA replication. We also demonstrated that copy numbers differ among different MAC chromosomes and that interchromosomal variations in copy number are consistent across individual cells. Our findings demonstrate that ddPCR can be used to model amitosis in T. thermophila and possibly in other ciliates.

Arrested crossover precursor structures form stable homologous bonds in a Tetrahymena meiotic mutant.

Meiotic DNA double-strand breaks produce reciprocally exchanged DNA strands, which mature into chiasmata that hold homologous chromosomes together as bivalents. These bivalents are subsequently separated in the first meiotic division. In a mutant lacking the newly identified Tetrahymena gene APRO1 (Anaphase promoting 1), meiosis is arrested by the end of prophase. Mature chiasmata are not formed but bivalents are connected via a molecular precursor structure. In-depth analysis of this arrested intermediate structure may help to elucidate the noncanonical molecular recombination pathway in Tetrahymena.

Identification and Characterization of Base-Substitution Mutations in the Macronuclear Genome of the Ciliate Tetrahymena thermophila.

Polyploidy can provide adaptive advantages and drive evolution. Amitotic division of the polyploid macronucleus (MAC) in ciliates acts as a nonsexual genetic mechanism to enhance adaptation to stress conditions and thus provides a unique model to investigate the evolutionary role of polyploidy. Mutation is the primary source of the variation responsible for evolution and adaptation; however, to date, de novo mutations that occur in ciliate MAC genomes during these processes have not been characterized and their biological impacts are undefined. Here, we carried out long-term evolution experiments to directly explore de novo MAC mutations and their molecular features in the model ciliate, Tetrahymena thermophila. A simple but effective method was established to detect base-substitution mutations in evolving populations whereas filtering out most of the false positive base-substitutions caused by repetitive sequences and the programmed genome rearrangements. The detected mutations were rigorously validated using the MassARRAY system. Validated mutations showed a strong G/C→A/T bias, consistent with observations in other species. Moreover, a progressive increase in growth rate of the evolving populations suggested that some of these mutations might be responsible for cell fitness. The established mutation identification and validation methods will be an invaluable resource to make ciliates an important model system to study the role of polyploidy in evolution.

Genomes: Programmed DNA Elimination in a Parasitic Nematode.

Programmed DNA elimination occurs in many eukaryotes. A new study provides a comprehensive view of programmed DNA elimination in a parasitic nematode, defining what sequences are eliminated from which chromosomal locations and presenting a new road map to investigate its molecular mechanism and evolution.

Non-coding RNA Transcription in Tetrahymena Meiotic Nuclei Requires Dedicated Mediator Complex-Associated Proteins.

To preserve genome integrity, eukaryotic cells use small RNA-directed mechanisms to repress transposable elements (TEs). Paradoxically, in order to silence TEs, precursors of the small RNAs must be transcribed from TEs. However, it is still poorly understood how these precursors are transcribed from TEs under silenced conditions. In the otherwise transcriptionally silent germline micronucleus (MIC) of Tetrahymena, a burst of non-coding RNA (ncRNA) transcription occurs during meiosis. The transcripts are processed into small RNAs that serve to identify TE-related sequences for elimination. The Mediator complex (Med) has an evolutionarily conserved role for transcription by bridging gene-specific transcription factors and RNA polymerase II. Here, we report that three Med-associated factors, Emit1, Emit2, and Rib1, are required for the biogenesis of small ncRNAs. Med localizes to the MIC only during meiosis, and both Med localization and MIC ncRNA transcription require Emit1 and Emit2. In the MIC, Med occupies TE-rich pericentromeric and telomeric regions in a Rib1-dependent manner. Rib1 is dispensable for ncRNA transcription but is required for the accumulation of double-stranded ncRNAs. Nuclear and sub-nuclear localization of the three Med-associated proteins is interdependent. Hence, Emit1 and Emit2 act coordinately to import Med into the MIC, and Rib1 recruits Med to specific chromosomal locations to quantitatively or qualitatively promote the biogenesis of functional ncRNA. Our results underscore that the transcription machinery can be regulated by a set of specialized Med-associated proteins to temporally transcribe TE-related sequences from a silent genome for small RNA biogenesis and genome defense.

Diversification of small RNA amplification mechanisms for targeting transposon-related sequences in ciliates.

The silencing of repetitive transposable elements (TEs) is ensured by signal amplification of the initial small RNA trigger, which occurs at distinct steps of TE silencing in different eukaryotes. How such a variety of secondary small RNA biogenesis mechanisms has evolved has not been thoroughly elucidated. Ciliated protozoa perform small RNA-directed programmed DNA elimination of thousands of TE-related internal eliminated sequences (IESs) in the newly developed somatic nucleus. In the ciliate Paramecium, secondary small RNAs are produced after the excision of IESs. In this study, we show that in another ciliate, Tetrahymena, secondary small RNAs accumulate at least a few hours before their derived IESs are excised. We also demonstrate that DNA excision is dispensable for their biogenesis in this ciliate. Therefore, unlike in Paramecium, small RNA amplification occurs before IES excision in Tetrahymena This study reveals the remarkable diversity of secondary small RNA biogenesis mechanisms, even among ciliates with similar DNA elimination processes, and thus raises the possibility that the evolution of TE-targeting small RNA amplification can be traced by investigating the DNA elimination mechanisms of ciliates.

Small RNA-Mediated trans-Nuclear and trans-Element Communications in Tetrahymena DNA Elimination.

Epigenetic inheritance of acquired traits is widespread among eukaryotes, but how and to what extent such information is transgenerationally inherited is still unclear. The patterns of programmed DNA elimination in ciliates are epigenetically and transgenerationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence and role of such small-RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is, indeed, determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small-RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small-RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination.

Whats, hows and whys of programmed DNA elimination in Tetrahymena.

Programmed genome rearrangements in ciliates provide fascinating examples of flexible epigenetic genome regulations and important insights into the interaction between transposable elements (TEs) and host genomes. DNA elimination in Tetrahymena thermophila removes approximately 12 000 internal eliminated sequences (IESs), which correspond to one-third of the genome, when the somatic macronucleus (MAC) differentiates from the germline micronucleus (MIC). More than half of the IESs, many of which show high similarity to TEs, are targeted for elimination in cis by the small RNA-mediated genome comparison of the MIC to the MAC. Other IESs are targeted for elimination in trans by the same small RNAs through repetitive sequences. Furthermore, the small RNA-heterochromatin feedback loop ensures robust DNA elimination. Here, we review an updated picture of the DNA elimination mechanism, discuss the physiological and evolutionary roles of DNA elimination, and outline the key questions that remain unanswered.

Negative Regulators of an RNAi-Heterochromatin Positive Feedback Loop Safeguard Somatic Genome Integrity in Tetrahymena.

RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.

A Zip3-like protein plays a role in crossover formation in the SC-less meiosis of the protist Tetrahymena.

When programmed meiotic DNA double-strand breaks (DSBs) undergo recombinational repair, genetic crossovers (COs) may be formed. A certain level of this is required for the faithful segregation of chromosomes, but the majority of DSBs are processed toward a safer alternative, namely noncrossovers (NCOs), via nonreciprocal DNA exchange. At the crossroads between these two DSB fates is the Msh4-Msh5 (MutSγ) complex, which stabilizes CO-destined recombination intermediates and members of the Zip3/RNF212 family of RING finger proteins, which in turn stabilize MutSγ. These proteins function in the context of the synaptonemal complex (SC) and mainly act on SC-dependent COs. Here we show that in the SC-less ciliate Tetrahymena, Zhp3 (a protein distantly related to Zip3/RNF212), together with MutSγ, is responsible for the majority of COs. This activity of Zhp3 suggests an evolutionarily conserved SC-independent strategy for balancing CO:NCO ratios. Moreover, we report a novel meiosis-specific protein, Sa15, as an interacting partner of Zhp3. Sa15 forms linear structures in meiotic prophase nuclei to which Zhp3 localizes. Sa15 is required for a wild-type level of CO formation. Its linear organization suggests the existence of an underlying chromosomal axis that serves as a scaffold for Zhp3 and other recombination proteins.

Heterochromatin aggregation during DNA elimination in Tetrahymena is facilitated by a prion-like protein.

Regulated aggregations of prion and prion-like proteins play physiological roles in various biological processes. However, their structural roles in the nucleus are poorly understood. Here, we show that the prion-like protein Jub6p is involved in the regulation of chromatin structure in the ciliated protozoan Tetrahymena thermophila Jub6p forms sodium dodecyl sulfate (SDS)-resistant aggregates when it is ectopically expressed in vegetative cells and binds to RNA in vitro Jub6p is a heterochromatin component and is important for the formation of heterochromatin bodies during the process of programmed DNA elimination. We suggest that RNA-protein aggregates formed by Jub6p are an essential architectural component for the assembly of heterochromatin bodies.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

Phosphorylation of an HP1-like protein is a prerequisite for heterochromatin body formation in Tetrahymena DNA elimination.

Multiple heterochromatic loci are often clustered into a higher order nuclear architecture called a heterochromatin body in diverse eukaryotes. Although phosphorylation of Heterochromatin Protein 1 (HP1) family proteins regulates heterochromatin dynamics, its role in heterochromatin bodies remains unknown. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation, and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, whereas it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination.

Phosphorylation of an HP1-like Protein Regulates Heterochromatin Body Assembly for DNA Elimination.

Heterochromatic loci are often assembled into higher-order heterochromatin bodies in diverse eukaryotes. However, the formation and biological roles of heterochromatin bodies are poorly understood. In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. Here, we show that the heterochromatin body component Jub1p promotes heterochromatin body formation and dephosphorylation of the Heterochromatin Protein 1-like protein Pdd1p. Through the mutagenesis of the phosphorylated residues of Pdd1p, we demonstrate that Pdd1p dephosphorylation promotes the electrostatic interaction between Pdd1p and RNA in vitro and heterochromatin body formation in vivo. We therefore propose that heterochromatin body is assembled by the Pdd1p-RNA interaction. Pdd1p dephosphorylation and Jub1p are required for heterochromatin body formation and DNA elimination but not for local heterochromatin assembly, indicating that heterochromatin body plays an essential role in DNA elimination.