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.

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.

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.

Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination.

Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms.

The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena.

Analysis of Piwi-loaded small RNAs in Tetrahymena.

Scan RNAs (scnRNAs) are developmentally regulated siRNAs of ~26-32 nucleotides in length that are involved in programmed DNA elimination in Tetrahymena. scnRNAs are loaded onto the Piwi-related protein Twi1p and 2'-O-methylated at their 3' termini. We describe two alternative strategies for analyzing the Twi1p-loaded scnRNAs: preparation of loaded scnRNAs by immuno-purification of the Twi1p-scnRNA complex and exclusion of non-methylated scnRNAs during cDNA library construction using periodate oxidation.

Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena.

The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.

The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.

Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

Two GW repeat proteins interact with Tetrahymena thermophila argonaute and promote genome rearrangement.

In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, approximately 28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of DeltaWAG1, DeltaCnjB, and double DeltaWAG1 DeltaCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.

Study of an RNA helicase implicates small RNA-noncoding RNA interactions in programmed DNA elimination in Tetrahymena.

Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and approximately 28-nucleotide small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p-scnRNA complexes identify micronuclear-limited sequences is not well understood. We show that a Twi1p-associated putative RNA helicase Ema1p is required for the interaction between Twi1p and chromatin. This requirement explains the phenotypes of EMA1 KO strains, including loss of selective down-regulation of scnRNAs homologous to macronuclear-destined sequences, loss of H3K9 and K27 methylation in the developing new macronucleus, and failure to eliminate DNA. We further demonstrate that Twi1p interacts with noncoding transcripts derived from parental and developing macronuclei and this interaction is greatly reduced in the absence of Ema1p. We propose that Ema1p functions in DNA elimination by stimulating base-pairing interactions between scnRNAs and noncoding transcripts in both parental and developing new macronuclei.

Peculiar behavior of distinct chromosomal DNA elements during and after development in the dicyemid mesozoan Dicyema japonicum.

The dicyemid mesozoans are obligate parasites that inhabit the cephalopod renal appendage. Dicyemids have a simple body, consisting of approximately 30 cells: one long cylindrical axial cell contains intracellular stem cells (called axoblast), from which embryos are derived, and is surrounded by some 30 peripheral somatic cells. Somatic cells divide at most eight times in their life span, and never divide after differentiation. During early somatic cell development, numerous unique DNA sequences are first amplified and then eliminated, in the form of extrachromosomal circular DNA, leading to genome reduction. In this study we demonstrate that the remaining sequences, single-copy genes and repetitive sequences, have very different fates. Single-copy genes, such as beta-tubulin, are initially amplified, presumably via endoreduplication, but subsequently decrease in copy number through development, suggesting that the whole genome is initially amplified and then the amplified DNAs are simply diluted in successive cell divisions, with little DNA replication. In contrast, repetitive sequences are maintained even in terminally differentiated somatic cell nuclei. Considering the increasing intensity of in-situ hybridization, incorporation of BrdU, and a general correlation between nuclear content and cell size, those repetitive sequences must be selectively endoreplicated in the peripheral cell nucleus, concomitant with the increase of cell size. The biological significance of this mechanism is discussed as a unique dicyemid adaptation to parasitism.

Differentiation of somatic mitochondria and the structural changes in mtDNA during development of the dicyemid Dicyema japonicum (Mesozoa).

Dicyemids (Mesozoa) are extremely simple multicellular parasites found in the kidneys of cephalopods. Their mitochondria are known to contain single-gene minicircle DNAs. However, it is not known if the minicircles represent the sole form of mitochondrial genome in these organisms. Here we demonstrate that high-molecular-weight (HMW) mtDNA is present in dicyemids. This form of mtDNA is probably limited to germ cells, and has been analyzed by PCR and Southern hybridization. In situ hybridization revealed that mtDNA is initially amplified during early embryogenesis, and then gradually decreases in copy number as larval development proceeds. Furthermore, we demonstrated using BrdU as a tracer that many of the mitochondria in terminally differentiated somatic cells no longer support DNA synthesis. Taking these observations into account, we propose an "amplification-dilution" model for mesozoan mtDNA. "Stem" mitochondria in the germ cells (1) amplify the HMW form of mtDNA in early embryos, followed by minicircle formation via DNA rearrangement, or (2) selectively replicate minicircles from the HMW DNA, concomitantly with the differentiation of the soma. Minicircle formation may itself lead to the loss of replication origins. Thereafter, the minicircles are simply distributed to daughter mitochondria without replication, resulting in the "somatic" mitochondria, which have lost the replicative form of the HMW mtDNA. The change in mtDNA configuration is discussed in relation to mitochondrial differentiation.

A "chimera" theory on the origin of dicyemid mesozoans: evolution driven by frequent lateral gene transfer from host to parasite.

The phylogenetic status of the enigmatic dicyemid mesozoans is still uncertain. Are they primitive multicellular organisms or degenerate triploblastic animals? Presently, the latter view is accepted. A phylogenetic analysis of 18S rDNA sequences placed dicyemids within the animal clade, and this was supported by the discovery of a Hox-type gene with a lophotrochozoan signature sequence. This molecular information suggests that dicyemid mesozoans evolved from an ancestral animal degenerately. Considering their extreme simplicity, which is probably due to parasitism, they might have come from an early embryo via a radical transformation, i.e. neoteny. Irrespective of this molecular information, dicyemid mesozoans retain many protistan-like or extremely primitive features, such as tubular mitochondrial cristae, endocytic ability from the outer surface, and the absence of collagenous tissue, while they do not share noticeable synapomorphy with animals. In addition, the 5S rRNA phylogeny suggests a somewhat closer kinship with protozoan ciliates than with animals. If we accept this clear contradiction, dicyemids should be regarded as a chimera of animals and protistans. Here, we discuss the traditional theory of extreme degeneration via parasitism, and then propose a new "chimera" theory in which dicyemid mesozoans are exposed to a continual flow of genetic information via eating host tissues from the outer surface by endocytosis. Consequently, many of their intrinsic genes have been replaced by host-derived genes through lateral gene transfer (LGT), implying that LGT is a key driving force in the evolution of dicyemid mesozoans.

Developmentally regulated extrachromosomal circular DNA formation in the mesozoan Dicyema japonicum.

The dicyemid mesozoans are simple multicellular parasites with a long cylindrical axial cell surrounded by a single outer layer of 20 to 30 ciliated peripheral somatic cells. Their larval development proceeds within the axial cell. Here we demonstrate the appearance of extrachromosomal circular DNAs and their fate during early embryogenesis in Dicyema japonicum. These DNAs are highly heterogeneous in sequence, suggesting that they consist of unique--not repetitive--elements. Potential open reading frames were not evident in the elements, so these DNAs are unlikely to have a protein-encoding function. In situ hybridization revealed that the circular DNA elements were restricted to the early embryonic larvae and gradually faded out as larvae approached maturity. Furthermore Southern blot analysis and polymerase chain reaction analysis using a high molecular weight DNA as a template provided evidence that the extrachromosomal DNA circles are originally present in chromosomes. These observations suggest DNA elimination--or selective replication--of the elements from chromosomes during early embryogenesis in dicyemid mesozoans.