Ciliated protozoa undergo extensive DNA rearrangements, including DNA elimination, chromosome breakage and DNA unscrambling, when the germline micronucleus produces the new macronucleus during sexual reproduction. It has long been known that many of these events are epigenetically controlled by DNA sequences of the parental macronuclear genome. Recent studies in some model ciliates have revealed that these epigenetic regulations are mediated by non-coding RNAs. DNA elimination in Paramecium and Tetrahymena is regulated by small RNAs that are produced and operated by an RNAi (RNA interference)-related mechanism. It has been proposed that the small RNAs from the micronuclear genome can be used to identify eliminated DNAs by whole-genome comparison of the parental macronucleus and the micronucleus. In contrast, DNA unscrambling in Oxytricha is guided by long non-coding RNAs that are produced from the somatic parental macronuclear genome. These RNAs are proposed to act as templates for the direct unscrambling events that occur in the developing macronucleus. The possible evolutionary benefits of these RNA-directed epigenetic regulations of DNA rearrangement in ciliates are discussed in the present chapter.
Recent advances in technology have enabled us to convert our somatic cells into pluripotent stem (iPS) cells, which can produce all types of cells in our body, by introducing a handful of transcription factors . This is possible because cells in our skin, muscle and brain are all derived from a single zygotic cell and thus have the same DNA. However, we can find an exception to this rule in the B- and T-cells in our blood; to recognize numerous different invaders such as viruses and bacteria, they have the ability to express over one million different receptors. Because humans have only ∼25000 genes, it is impossible to express such varieties of receptors even if all genes are allocated to their production. Instead, immune cells produce multiple different receptor-coding genes by making DNA rearrangements. For example, the human Ig (immunoglobulin) heavy chain locus in the germline has ∼50 VH, 27 DH and 6 JH encoding genes; one of each regional gene is recombined by DNA rearrangement during B-cell maturation to generate ∼8000 different Ig heavy chains. Similar DNA rearrangements produce ∼200 Ig light chains from a germline light-chain locus. Because Ig consists of a heavy chain and a light chain, a repertoire of ∼1.6 million (=8000×200) antibodies can be produced .
Similarly, many different eukaryotes undergo a variety of DNA rearrangements during certain developmental processes, such as mating-type switching in yeast and chromatin diminution in the somatic cell lineages of parasitic nematodes and crustaceans . Additionally, elimination of entire chromosomes has been observed in somatic cells of early embryos of hagfish and sciarid flies . Therefore developmentally regulated DNA rearrangements are widespread among eukaryotes. One of the most extreme examples of DNA rearrangement has been observed in the ciliated protozoa. Recent studies have revealed that some types of DNA rearrangements in ciliates are epigenetically regulated by small and/or long non-coding RNAs. In this chapter, I focus on these epigenetic regulations of DNA rearrangements in ciliates. For recent advancements on the molecular mechanisms controlling DNA rearrangements in ciliates, please see other reviews [5,6].
DNA rearrangement in ciliated protozoa
Ciliates are a group of primarily single-celled eukaryotes common almost everywhere there is water. They are similarly evolutionarily distant from Unikonts (including animals, fungi and amoebas) and Plantae (including algae and land plants), and belong to another supergroup, Chromalveolata. Most ciliates show nuclear dimorphism by the presence of a germline micronucleus and a somatic macronucleus (Figure 1A). Only the micronucleus has the ability to undergo meiosis, which, followed by fertilization, forms the zygotic nucleus. The zygotic nucleus produces new micro- and macro-nuclei for the next generation. DNA rearrangements occur during the differentiation from the zygotic nucleus to the new macronucleus (Figure 1A). In this process, approx. 20% of the micronuclear DNA is deleted in the oligohymenophorean ciliates Paramecium and Tetrahymena, whereas more than 95% of the genome is eliminated in some spirotrich ciliates, such as Euplotes, Stylonychia and Oxytricha . Endoreplication of macronuclear chromosomes also occurs during macronuclear development. The degree of endoreplication is greater in spirotrichs than oligohymenophoreans. For example, Tetrahymena has ∼50 copies of macronuclear chromosomes, whereas Oxytricha has ∼2000 copies .
A variety of DNA rearrangements has been reported in several different ciliates and I classify them here into four types. A Type I rearrangement is defined by the elimination of internal DNA segments followed by ligation of the two flanking macronuclear-destined sequences (Figure 1B, I). The eliminated DNAs vary in length (∼30 bp to ∼20 kbp) and in their sequence nature (single-copy sequences, transposons and other repeated sequences). Some of the internal DNA segments are precisely excised at the nucleotide level, whereas others are imprecisely eliminated with variable boundaries. These are called IESs (internal eliminated sequences; although only the precisely excised single-copy sequences are called IESs in Paramecium, I call all internally eliminated sequences IESs in this chapter). A Type II rearrangement is similar to a Type I rearrangement but leads to chromosome fragmentation followed by the addition of telomeres (Figure 1B, II). A Type III rearrangement involves chromosome breakage at the Cbs (chromosome breakage sequence) site accompanied by short DNA trimming and de novo telomere formation (Figure 1B, III). Finally, Type IV rearrangements involve ‘unscrambling’ of protein-encoding DNA segments (Figure 1B, IV). In this process, ‘scrambled’ protein-encoding sequences in the micronucleus are joined and assembled (scrambled) into the proper order in the macronucleus. Not all types of DNA rearrangements can be observed in a single ciliate species. For example, Tetrahymena thermophila show only Type I and III rearrangements, and Type IV rearrangements have been detected only in some spirotrich ciliates. The types of DNA rearrangements observed in different ciliate species are summarized in Figure 1(C).
As a result of these DNA eliminations and fragmentations, Paramecium and Tetrahymena produce macronuclear chromosomes with an average size of ∼300 kbp and ∼800 kbp respectively . On the other hand, spirotrich ciliates have ∼20000 extensively fragmented macronuclear chromosomes with an average size of ∼2 kb. They often contain only single genes and are referred to as ‘gene-sized’ chromosomes .
Epigenetic effects of parental macronuclear sequences on DNA rearrangement in the new macronucleus
Several lines of evidence suggest that there are epigenetic mechanisms that use the primary sequence of the parental macronucleus to direct DNA rearrangements in the developing macronucleus.
The first case of such an epigenetic effect on DNA rearrangement was uncovered through the analysis of the d48 mutant of Paramecium tetraurelia. In wild-type cells, Type II DNA rearrangements occur proximal to the A-type surface antigen gene (A-gene), which is retained in the macronucleus (Figure 2A). The d48 mutant has a deletion of the A-gene in the macronucleus, while the micronuclear A-gene remains intact. In the sexual progeny of d48 mutants, the A-gene is eliminated from the new macronucleus, and thus the d48 mutation is epigenetically inherited in the next generation (Figure 2B) . Moreover, microinjection of DNA fragments containing the A-gene into the parental macronuclei of d48 mutants can induce retention of the A-gene in the new macronuclei (Figure 2C) [9,10]. Epigenetic effects in DNA rearrangements are also seen when IESs are introduced into the parental macronuclei of wild-type cells. In Paramecium, injection of a plasmid containing an IES in the G surface antigen gene into the macronucleus causes retention of this IES in the newly formed macronucleus (Figure 2D) . Similar epigenetic effects in IES eliminations were observed in Tetrahymena . These phenomena indicate that both Type I and II rearrangements (DNA elimination) can be influenced by the DNA sequence of the parental macronucleus, such that the new macronucleus copies the sequence pattern of the parental macronucleus. To achieve these maternal regulations, some sequence-specific information must be transferred from the parental macronucleus to the new macronucleus.
Small RNA-directed DNA rearrangement
The finding that the micronuclear genome is bi-directionally transcribed during conjugation in Tetrahymena  provided the first indication that micronuclear RNAs could play a role in the maternal regulation of DNA rearrangements. Although the micronucleus is transcriptionally inert in most life stages, an exception to this has been observed during prophase meiosis in Tetrahymena . It was determined that these micronuclear RNAs are processed to ∼28–29 nucleotide small RNAs, called scn (scan) RNAs, by the Dicer-like protein Dcl1p [15,16] and then form complexes with the Argonaute protein Twi1p [17,18]. Dcl1p and Twi1p are essential for the production and stable accumulation of scnRNAs respectively, and for Type I rearrangements (IES elimination) [15–17]. Dicer and Argonaute family proteins are conserved core components of the RNAi (RNA interference)-machinery and are involved in post-transcriptional as well as transcriptional gene silencing in many eukaryotes . Dicer-dependent accumulation of scnRNAs and its requirement in DNA rearrangements (Type I and II) has also been reported in Paramecium . Therefore scnRNAs derived from micronuclear transcripts play a pivotal role in DNA elimination in oligohymenophoreans in a manner similar to RNAi-directed transcriptional gene silencing in other eukaryotes.
The results above suggest that scnRNAs act as messengers that transmit maternal information from the parental macronucleus to the new macronucleus. How can they direct precise DNA rearrangements? In Tetrahymena, although scnRNAs complementary to both the macronuclear-destined sequences and the micronuclear-specific sequences (mostly IESs in Tetrahymena) are produced in the early conjugation stage, only the latter scnRNAs become gradually enriched in the mid-conjugation stages [18,21]. In the mid-conjugation stages, the scnRNA–Twi1p complex is localized to the parental macronucleus. Later, the complex, including scnRNAs enriched in IES sequences, is transported into the new macronucleus. Therefore it is reasonable to speculate that some mechanism specifically degrades scnRNAs complementary to the genomic DNA in the parental macronucleus, and this could be the basis of the maternal regulation of DNA rearrangements (Figure 3) .
The molecular mechanism that induces selective degradation of scnRNAs complementary to the macronuclear genome is still unclear. Our own study in Tetrahymena  suggested that nascent non-coding transcripts mediate the interaction between chromatin and scnRNA–Twi1p complexes in the parental macronucleus and that this interaction is dependent on the putative RNA helicase Ema1p. Moreover, EMA1-knockout strains have defects in the selective degradation of scnRNAs and in DNA elimination. Therefore we have proposed that scnRNA degradation is induced by a base-pairing interaction between scnRNAs and nascent non-coding transcripts from the parental macronucleus. The importance of the parental macronuclear transcripts for proper IES elimination has also been demonstrated in studies of another ciliate, Paramecium tetraurelia . Down-regulation of non-coding transcripts from the parental macronucleus by an RNAi technique blocked the scanning process in the targeted regions and induced ectopic DNA elimination.
Methylation of histone H3 at lysine residues 9 (H3K9me) and 27 (H3K27me) and the chromodomain protein Pdd1p, which binds to these modifications, specifically accumulate on IESs and are required for DNA elimination . Therefore IESs are wrapped in heterochromatin structure before they are eliminated. Since disruption of TWI1 inhibits H3K9me accumulation , the RNAi-related pathway is upstream of heterochromatin formation.
RNA-directed DNA unscrambling in Oxytricha
Type IV DNA rearrangements (DNA unscrambling) in the spirotrich ciliate Oxytricha are also regulated by non-coding RNAs. Oxytricha predominantly utilizes long RNAs to guide the unscrambling event (Figure 4A), although some role of small RNAs in this rearrangement has been suggested . The long RNA-guided DNA unscrambling was first proposed as a theoretical model  and recently demonstrated experimentally . Bi-directional transcription of the parental macronuclear genome occurs in early conjugation in Oxytricha. Spirotrich ciliates have very short ‘gene-sized’ macronuclear chromosomes, and the macronuclear long RNAs are probably produced by ‘telomere to telomere’ transcription (Figure 4A). Disruption of specific parental macronuclear long RNAs by RNAi inhibits unscrambling of the corresponding loci in the new macronucleus (Figure 4B), and injection of artificial RNAs reprogrammes the unscrambling orders (Figure 4C). Therefore Type IV DNA rearrangements in Oxytricha are epigenetically regulated by the DNA sequence of the parental macronuclear genome, and long macronuclear RNAs act as templates in these events (Figure 4A). Moreover, the occasional transfer of point mutations from RNA templates to the new macronuclear DNAs has been observed . This indicates that there is an RNA-directed proofreading mechanism. Although the molecular mechanisms involved in RNA-guided DNA chromosome rearrangements and RNA-directed proofreading are currently unclear, future studies on these phenomena would provide us clear examples about how acquired somatic mutations can be transmitted to the next generation without altering the germline genome.
Evolutionary advantages of epigenetically regulated DNA rearrangement
Epigenetic regulation of DNA rearrangements by macronuclear DNA sequences must have some evolutionary advantage for the survival of ciliates. Three potential benefits of these events can be proposed.
The first advantage is sequence-independent recognition of molecular parasites such as viruses and transposons. Because many of the eliminated sequences during Type I and II DNA rearrangements are related to transposons, it is thought that one of the major roles of DNA rearrangements in ciliates is the elimination of transposons from the transcriptionally active macronucleus. Indeed, exogenous sequences experimentally introduced into the micronucleus are eliminated by Type I rearrangement during macronuclear development [28,29]. This epigenetic mechanism could target any type of DNA inserted at any locus of the micronucleus for elimination.
The second advantage is the inheritance of acquired somatic mutations in the next sexual generations. Because the macronucleus is polyploid and its chromosomes are amitotically segregated during vegetative growth, mutations in the macronuclear chromosomes can be assorted positively or negatively. If a mutation is beneficial to cells, it can be fixed in a population during vegetative growth. The epigenetic regulation of the DNA rearrangements described above could act as a mechanism that transmits beneficial somatic mutations to the next generation. Although only large deletions in the macronucleus can be inherited in the next sexual generations by the small RNA-directed DNA rearrangement system in the oligohymenophorean ciliates Paramecium and Tetrahymena, RNA-guided DNA rearrangements in Oxytricha can result in the transmission of translocations and even point mutations to the next generations.
The third advantage is the inheritance of beneficial variants among alternatively rearranged forms of a gene. DNA rearrangements can produce variants of macronuclear chromosomes from a single micronuclear genome by alternative rearrangements. If some specific variant (or combinations of variants) is advantageous to the growth and/or survival of cells, it would dominate over other variants, as in the mechanism described above. Once some variant is fixed in the macronucleus, the pattern of DNA rearrangement in the next sexual generation can be biased toward this variant form by the epigenetic mechanism. Also, because the micronucleus retains the original genome, cells have a chance to quickly return to an unbiased rearrangement when the variant loses its advantage for survival owing to some environmental change.
Although the epigenetic regulation of DNA rearrangements in the ciliated protozoa has been known for a long time, its molecular basis has only recently begun to be revealed. Small RNA-directed DNA elimination in the oligohymenophorean ciliates Paramecium and Tetrahymena and long RNA-guided DNA unscrambling in the spirotrich ciliate Oxytricha were discussed in this chapter. How these seemingly distinct mechanisms have evolved in different classes of ciliates is unclear. Future studies on other ciliate classes may help to solve this issue. Additionally, the detailed molecular mechanisms regulating these events, such as the biogenesis and selection of scnRNAs in Tetrahymena and Paramecium, the transcription of long template RNAs in Oxytricha and the induction of DNA rearrangements by these non-coding RNAs, are still mostly unknown. I expect established genetic tools in these model ciliates, such as gene knockout and RNAi techniques, and fully sequenced genomes will help to answer these unsolved questions in the near future.
• Several different types of DNA rearrangements are observed in ciliated protozoa.
• DNA elimination (Type I and II DNA rearrangement) and DNA unscrambling (Type IV DNA rearrangement) occur in the newly developed macronucleus and are epigenetically regulated by the primary DNA sequences of the parental macronucleus.
• DNA eliminations in the oligohymenophorean ciliates Paramecium and Tetrahymena are directed by small non-coding RNAs produced by an RNAi-related mechanism.
• DNA unscrambling in the spirotrich ciliate Oxytricha is guided by long macronuclear non-coding RNAs.
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