Small RNAs in genome rearrangement in Tetrahymena

Small RNAs in genome rearrangement in Tetrahymena Kazufumi Mochizuki and Martin A Gorovsky
Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes. Addresses Department of Biology, University of Rochester, Hutchison Hall 425, Rochester, New York 14627, USA
e-mail: [email protected]

tion required for vegetative cell growth occurs in the macronucleus. Most ciliates studied to date undergo extensive genome rearrangements during conjugation (see [2,3]). In Tetrahymena, programmed genome rearrangements result in elimination of 15% of the genome, while the rest of the genome is endoreplicated 50 times. Two types of genome rearrangement occur. The first is deletion of internal eliminated sequences (IESs), accompanied by ligation of the flanking macronucleus-destined sequences (MDSs) (Figure 1a). The 6000 IESs in Tetrahymena vary from 0.5 to >20 kb in length. Excision of IESs can occur reproducibly at a specific site or at a limited number of alternative sites. The second type of rearrangement involves chromosome breakage followed by small (<50 bp) deletions of breakage eliminated sequences (BESs) and addition of telomeres (Figure 1b). This produces 2–300 macronuclear chromosomes from the five chromosomes in the micronuclear (haploid) genome. BESs also occur at precise locations.

Current Opinion in Genetics & Development 2004, 14:181–187 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Stephen D Bell and Andy Bannister 0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.01.004

Abbreviations BES breakage eliminated sequence Cbs chromosome breakage sequence dsRNA double stranded RNA H3K9 Lysine 9 of histone H3 IES internal eliminated sequence MDS macronucleus destined sequence PPD PAZ and Piwi domain RNAi RNA interference scnRNA scan RNA

Although the IESs and BESs in Tetrahymena (and other ciliates) have been known for some time, two long-standing issues remained unresolved. The first is how IESs are recognized. BESs contain a highly conserved, 15 bp chromosome breakage sequence (Cbs), a likely site for recognition by (unknown) proteins [4]. By contrast, no shared common sequences were identified in IESs. The other issue concerns the biological function and selective advantage of the genome rearrangements. Endoreplication of the genome is likely to be required for the large cell size typical of ciliates that probably enables their remarkable intracellular diversity and enhances their ability to feed. But why DNA elimination and chromosome fragmentation evolved in ciliates was not clear. In this review, we describe the recent discovery of the involvement of an RNAi-related mechanism in the genome rearrangement of Tetrahymena that helps explain how IESs are recognized and provides a likely function for DNA elimination. The function of chromosome fragmentation remains obscure.

Introduction Like most ciliated protozoans, Tetrahymena thermophila (referred to as Tetrahymena in this review) have two structurally and functionally different nuclei in a single cell [1]. The diploid, germline micronucleus and the polyploid, somatic macronucleus are derived from the same zygotic nucleus formed by fertilization of two micronucleus-derived, haploid, meiotic nuclei during the sexual process of conjugation [1]. Concomitant with formation of a new macronucleus during conjugation, the old macronucleus is destroyed. Most, if not all, transcripwww.sciencedirect.com

An RNAi machinery is required for genome rearrangement in Tetrahymena The PPD protein family — also called the Argonaute or AGO1/Piwi-related family — contains PAZ and Piwi domains. PAZ domains have recently been shown to bind RNA [5–7]. The function of Piwi domains is unknown. PPD proteins are involved in RNAi-related, post-transcriptional and/or transcriptional gene silencing in many eukaryotes although their exact role is not well understood [8]. Current Opinion in Genetics & Development 2004, 14:181–187

182 Chromosomes and expression mechanisms

Figure 1

(a) IES Micronucleus

Macronucleus BES

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Cbs Micronucleus

cleus-specific IES sequences were detected during conjugation [13] makes it likely that micronucleus-derived dsRNAs are available to be processed into the small RNAs. When radioactive small RNAs were used to probe macronuclear and micronuclear DNAs, they hybridized preferentially to micronuclear DNA, indicating that these RNAs are preferentially derived from micronucleus-specific sequences. Strikingly, in cells with disrupted TWI1 genes, the amount of the small RNAs was greatly reduced [9]. Thus, Twi1p is required for production or stability of the small RNAs. These observations strongly implicate an RNAi-like process in IES elimination.

Macronucleus Telomeres Current Opinion in Genetics & Development

Two types of genome rearrangement in Tetrahymena. (a) IES elimination. A part of the micronuclear chromosome (IES, green) is eliminated (dotted lines) and macronucleus destined sequences (MDSs, black) are re-ligated. (b) Chromosome breakage. A part of the micronuclear chromosome (BES, blue) is eliminated and telomeres (red circles) are added to the ends of MDSs (black). BES contains a conserved 15 bp sequence, Cbs (pink), that is the site of chromosome breakage.

Twi1p, a Tetrahymena PPD protein, is specifically expressed during conjugation [9], appearing first in the cytoplasm of early conjugating cells, then accumulating in the parental macronucleus. It is lost from the old macronucleus and simultaneously accumulates in the new macronucleus when differentiation initiates, and later disappears. When the TWI1 gene, encoding Twi1p, was disrupted, cells completed conjugation, but none of the progeny survived [9]. In these progeny, IES elimination was not observed and BES elimination was also severely affected [9]. This lethality was probably caused by the defects in genome rearrangement, as other genes that prevent IES excision show a similar phenotype. The requirement for Twi1p suggested that an RNAi-related mechanism is involved in genome rearrangement in Tetrahymena.

Small RNAs homologous to IESs are expressed during conjugation In Tetrahymena, small (28 nucleotide) RNAs appeared shortly after conjugation began and were detectable until late conjugation when genome rearrangement occurred [9]. Though slightly longer than small RNAs observed in RNAi-related pathways, their 50 -phosphate and 30 hydroxyl ends [9] suggest they were processed by a Dicer-like RNase III activity as in RNAi [10]. Dicer homologues have been identified in Tetrahymena and their functions are under investigation. The observations that transcription occurred in the micronucleus early during conjugation, but not during vegetative growth [11,12], and that bi-directional transcripts containing micronuCurrent Opinion in Genetics & Development 2004, 14:181–187

Epigenetic effects of parental macronuclear sequences on IES formation in new macronuclei Before Twi1p and small RNAs were implicated in IES elimination, evidence suggested that there was an epigenetic mechanism that uses the primary sequence itself of the parental macronucleus to specify the IESs of the developing new macronucleus (reviewed in [2,3,14]). In Tetrahymena, introduction of DNA containing an IES sequence into the parental macronucleus specifically inhibits DNA elimination of that IES when the new macronucleus forms (Figure 2b) [15]. In another ciliate, Paramecium, an IES in the G surface antigen gene that had somehow been retained in the macronucleus inhibited elimination of this IES when the new macronucleus formed [16] (Figure 2b), and deletion of another (A-type) surface antigen gene in the macronucleus resulted in deletion of this locus in the new macronucleus, even though the micronuclear A-antigen gene was intact (Figure 2c) [17]. These phenomena indicated that sequence-specific information could be transferred from the parental to the new macronucleus. The discovery of small, IES-related RNAs made them the most likely candidate for a role in this transfer. Interestingly, the pattern of transfer of epigenetic information between the old and new macronucleus also parallels the transfer of Twi1p between these two types of nuclei.

The scan RNA model for IES elimination We proposed the scan RNA model (Figure 3) to explain how IESs can be eliminated in the absence of consensus DNA sequences by an RNAi-related mechanism that accounts for the observed epigenetic regulation [9]. In this model, the whole micronuclear genome or regions around IESs are first transcribed bi-directionally in early conjugation. These transcripts then form dsRNAs that are processed to small RNAs by an RNAi-like machinery. The site of formation of these small RNAs (micronucleus, cytoplasm, or macronucleus) is unknown. We call these scan RNAs (scnRNAs) because we believe they scan macronuclear and micronuclear DNAs to determine the identity of IESs (and possibly BESs). The scnRNAs then www.sciencedirect.com

Small RNAs in genome rearrangement in Tetrahymena Mochizuki and Gorovsky 183

Figure 2

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MDS Current Opinion in Genetics & Development

Epigenetic control of IES elimination by the old macronucleus. Cells before conjugation are on the left and cells post-conjugation are on the right. (a) Genome rearrangement in a wild-type strain. IES-A (dark green) and IES-B (yellow) are eliminated during new macronucleus development, while MDS (red) is retained. (b) Effect of an IES in the old macronucleus. When IES-A is artificially or accidentally introduced into the old macronucleus, elimination of that IES is inhibited in the new macronucleus. (c) Effect of the absence of MDS in the old macronucleus. When MDS is accidentally lost in the old macronucleus, MDS is also eliminated in the new macronucleus. Mac, macronucleus; Mic, micronucleus.

accumulate in the (old) macronucleus, probably in association with Twi1p, where we propose that those having a homologous macronuclear DNA sequence are degraded. As a result, only scnRNAs homologous to micronucleusspecific sequences remain in the old macronucleus. Finally, we propose that these micronucleus-specific scnRNAs move (again, probably in association with Twi1p) to the developing new macronucleus. There, the sequences homologous to the scnRNAs are identified and targeted for elimination. Chromosome breakage probably occurs by recognition of the conserved 15 bp Cbs sequence. However, in cells with disrupted TWI1 [9] or PDD2 (another gene required for genome rearrangement [18]), both IES and BES elimination were affected. Thus, scnRNAs may function to determine BESs as well as IESs. www.sciencedirect.com

Tests of the scan RNA model Recent studies have tested some of the predictions of the scnRNA model. The likely re-localization of small RNAs from old to new macronuclei in association with Twi1p is supported by the finding that the small RNAs can be coimmunoprecipitated with Twi1p at stages of conjugation when most of the Twi1p is in the old or in the new macronucleus (K Mochizuki, MA Gorovsky, unpublished). The predicted enrichment of IES sequences in scnRNAs as conjugation proceeds has been demonstrated by showing that the ratio of the amount of the small RNAs hybridizing to micronuclear versus macronuclear DNA increases as conjugation progresses, until new macronuclei have developed (K Mochizuki, MA Gorovsky, unpublished observations). Current Opinion in Genetics & Development 2004, 14:181–187

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Figure 3

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viruses and transposons that might be harmless when in the transcriptionally inactive micronucleus, but could produce detrimental phenotypes in the transcriptionally active macronucleus. When heterokaryons were created with the bacterial transposon Tn5 neomycin resistance gene introduced at several different sites in the micronucleus, but not the (old) macronucleus, this gene was eliminated from the new macronucleus formed after conjugation, just like IESs ([19]; Y Liu, X Song, MA Gorovsky, KM Karrer, unpublished). Thus, this defense system is active and easily demonstrated in Tetrahymena. A further implication of these studies is that micronuclear transcription leading to the formation of scnRNAs is likely to be highly promiscuous.

Genome rearrangement and heterochromatin formation are highly related 4 New Mac

Current Opinion in Genetics & Development

The scanning model for the role of small RNAs in IES elimination. Step1: the genome of the micronucleus (Mic) including IES sequences is transcribed bi-directionally and these transcripts form dsRNA. Step2: the dsRNAs are processed to the small RNA (named scnRNA) by a Dicer-like RNase III. Step3: the scnRNAs are transferred to the old macronucleus (Old Mac) and any scnRNAs homologous to DNAs in the old macronucleus are degraded. The dotted line indicates the position of IESs that were eliminated during formation of the Old Mac in the preceding conjugation. Step 4: scnRNAs that are not degraded in the old macronucleus (those homologous to IESs) are then transferred to the developing new macronucleus (New Mac) where they target IESs to be eliminated by base pairing.

A recent study [19] provides a striking demonstration of the involvement of an RNAi-related mechanism in IES elimination. When in vitro transcribed dsRNA homologous to an MDS sequence was injected into conjugating cells, this sequence was eliminated in the newly formed macronucleus like an IES [19]. Thus dsRNA, by itself, can serve as a signal for changing the identification of an MDS into an IES without introducing any IES-specific sequences into the micronucleus. Efficiency of elimination of their homologous sequences increased when dsRNAs were injected at later stages [19]. We speculate that early injection overloads and later injection bypasses the scanning process in the old macronucleus.

IES elimination can serve as a defense system against foreign DNAs Recent studies provide additional insight into the mechanism of how IESs originate and strongly support the hypothesis that IES elimination can function as a defense system against invading genetic elements such as Current Opinion in Genetics & Development 2004, 14:181–187

The process of IES elimination bears striking similarity to the formation of heterochromatin in other eukaryotes. Most IESs are repeated sequences and some show homology with transposons [2,3]. Thus, IESs are similar to sequences found in heterochromatin in other eukaryotes. During the process of IES elimination, these sequences are found in dense, heterochromatin-like, regions [20]. Recent studies in many eukaryotes have shown that heterochromatin formation is accompanied by histone hypoacetylation, requires methylation of histone H3 at residue K9 (H3K9) and accumulation of chromodomain proteins [21]. In Tetrahymena, IES elimination can be inhibited by treatment with an inhibitor of histone deacetylase [22] and the only place that H3K9 methylation is detectable is in association with IESs during macronuclear development [23]. In Schizosaccharomyces pombe, H3K9 methylation is required to initiate heterochromatin formation; and in Tetrahymena, the replacement of H3K9 by glutamine eliminates methylation and inhibits IES elimination [24]. Chromodomain proteins (Pdd1p and Pdd3p), which are known to bind to methylated H3K9, also are associated with IESs [23,25] during genome rearrangement. Disruption of the PDD1 gene prevents IES elimination [26] and ectopic localization of Pdd1p using a LexA–Pdd1p fusion protein and the DNA sequence for LexA binding promotes sequence elimination [23]. These observations suggest that genome rearrangement in Tetrahymena is closely related to heterochromatic gene silencing.

An RNAi-related mechanism controls heterochromatin formation and IES elimination Disruption of TWI1 prevented H3K9 methylation during development of the new macronucleus [24]. Thus, an RNAi-related mechanism is required for the formation of the heterochromatin-like state that precedes IESs elimination in Tetrahymena. The RNAi machinery is also required for heterochromatin formation followed by transcriptional gene silencing in other eukaryotes. In S. pombe, www.sciencedirect.com

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Ago1 (a PPD protein), Dcr1 (Dicer homologue) and Rdp1 (RNA-dependent RNA polymerase 1) are required for methylation of H3K9 and gene silencing in centromeric regions and mating type loci [27,28], and small RNAs homologous to centromeric repeats have been isolated [29]. In a plant, Arabidopsis thaliana, AGO4 (a PPD protein) and SDE4 are required for H3K9 (and DNA) methylation on a retroelement and for accumulation of small RNAs homologous to it [30,31]. In Drosophila melanogaster, small RNAs homologous to transposons, satellite and microsatellite DNAs and Su(Ste) repeats have been cloned [32]. These observations strongly suggest that the involvement of the RNAi-related

mechanisms in heterochromatic gene silencing is evolutionarily conserved among eukaryotes. Genome rearrangement in Tetrahymena and heterochromatin formation are illustrated in Figure 4. The biggest difference between them is the end point of the process: ciliates eliminate the heterochromatinized sequences instead of maintaining the transcriptionally silenced heterochromatin state. Considered in this way, IES elimination can be viewed as an ultimate form of gene silencing. Thus, heterochromatinization in diverse organisms appears to be quite similar, up to and including the process of H3K9 methylation and involvement of chromodomain

Figure 4

Bi-directional transcripts from IESs or heterochromatic sequences

siRNAs

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Me CMe C MeCMeMe CMe Maintenance of silencing, DNA methylation Current Opinion in Genetics & Development

Genome rearrangement and heterochromatin formation. In both IES elimination in Tetrahymena and heterochromatin formation in S. pombe and Arabidopsis, bi-directional transcription occurs on the targeted sequences. These transcripts likely form dsRNAs that are processed to the siRNAs by Dicer-related RNases. Twi1p in Tetrahymena and probably Ago1 in fission yeast and AGO4 in Arabidopsis associate with the small RNAs. These small RNAs identify IESs or heterochromatic sequences. Then, H3K9 histone methyltransferase (HMT) methylates histone H3. Chromodomain proteins can bind to the methylated H3K9 and/or to RNA. The H3K9-specific HMT may itself be a chromodomain protein or may bind to the chromodomain proteins that accumulate on the sequences recognized by the small RNAs. Thus the H3K9 HMT and chromodomain proteins can interact in a positive loop to spread, maintain and replicate heterochromatin. Heterochromatin is maintained by this positive loop in S. pombe and Arabidopsis. In Arabidopsis, DNA methylation also occurs and probably contributes to the establishment and maintenance of heterochromatin. In many organisms, heterochromatinization is a metastable state that can occasionally revert to euchromatin. By contrast, in Tetrahymena, the heterochromatinized IESs are eliminated and cannot be reactivated. The striped line represents the IES or heterochromatic sequence. The black line represents MDS or non-heterochromatic sequence. The red, wavy lines represent RNA. www.sciencedirect.com

Current Opinion in Genetics & Development 2004, 14:181–187

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proteins. However, the steps that follow in different organisms can differ from recruitment of additional proteins that spread the heterochromatic state (S. pombe), to recruitment of enzymes involved in DNA elimination (ciliates) or DNA methylation (Arabidopsis).

Conclusions IES elimination, the most striking and heretofore the most perplexing feature of Tetrahymena genome rearrangement, is now known to involve an RNAi-related mechanism and is similar to heterochromatin formation in other eukaryotes. The small RNAs likely mediate the epigenetic effect of the old macronuclear genome on IES elimination in the new macronucleus by identifying the micronucleus-specific sequences. This system not only ensures accurate genome rearrangement, but also works as a defense against deleterious genetic elements that invade the germline. Although the broad outlines of heterochromatinization and of IES elimination are emerging, much remains unknown. Very little is known about the initial stages of production and amplification of the dsRNAs that initiate these processes. Although PPD proteins are clearly involved in RNAi production and/or accumulation, the detailed function of even their defining (PAZ and PIWI) domains are unknown. In Tetrahymena, nothing is known about the nature or recruitment of the enzymes to heterochromatinized IESs to effect their elimination.

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29. Reinhart BJ, Bartel DP: Small RNAs correspond to centromere heterochromatic repeats. Science 2002, 297:1831. 30. Zilberman D, Cao X, Jacobsen SE: ARGONAUTE4 control of  locus-specific siRNA accumulation and DNA and histone methylation. Science 2003, 299:716-719. AGO4, a PPD protein, is required for H3K9 and DNA methylation followed by silencing of the SUP gene. Furthermore, mutation of AGO4 blocks H3K9 and DNA methylation and accumulation of 25 nucleotide siRNAs that corresponds to a retroelement, AtSN1. These results suggest that long siRNA produced by an RNAi-like machinery is required for heterochromatin formation in Arabidopsis. 31. Hamilton A, Voinnet O, Chappell L, Baulcombe D: Two classes  of short interfering RNA in RNA silencing. EMBO J 2002, 21:4671-4679. Two classes of siRNA, short (21–22 nucleotides) and long (24–26 nucleotides), are produced from a GFP transgene in Arabidopsis. Using mutations in the RNAi pathway and viral suppressors of RNAi, they demonstrated that the shorter siRNAs correlate with mRNA degradation and longer siRNAs are involved in systemic silencing and DNA methylation of the homologous sequence. 32. Aravin AA, Lagos-Quintana M, Yalcin A, Zavolan M, Marks D,  Snyder B, Gaasterland T, Meyer J, Tuschl T: The small RNA profile during Drosophila melanogaster development. Dev Cell 2003, 5:337-350. Out of 560 small RNAs with characteristics of RNase III products isolated from Drosophila, 178 are homologous to repeated sequences such as transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs are involved in heterochromatin formation in Drosophila.

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