Large-scale genome remodelling by the

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Research in Microbiology 155 (2004) 399–408

Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium Mireille Bétermier CNRS UMR 8541, Laboratoire de Génétique Moléculaire, École Normale Supérieure, 46, rue d’Ulm, 75005 Paris, France Received 23 October 2003; accepted 20 January 2004 Available online 22 March 2004

Abstract In Paramecium, during the development of the somatic macronucleus, precise excision of thousands of single-copy non-coding sequences is initiated by specific DNA double-strand breaks, while imprecise elimination of germ-line-limited repeated sequences leads to internal deletions or chromosome fragmentation. Recent data point to a role of non-coding RNAs in the epigenetic programming of these rearrangements.  2004 Elsevier SAS. All rights reserved. Keywords: DNA excision; Epigenetic control; Double-strand break; IES

1. Introduction Site-specific DNA deletion systems typically involve the sequence-specific binding of a protein complex to nucleotide sequence motifs, generally 10–20 bp long and located close to the ends of the deleted fragment, and the cleavage of DNA at the two sites that define the deletion boundaries (reviewed in [7]). The accessibility of these recombination loci may be regulated—positively or negatively—by additional factors, such as transcription and/or the chromatin structure of the rearranged region, or the methylation state of its DNA. Activation of DNA cleavage may be regulated further by the topologically correct alignment of the two ends of the deleted fragment within a so-called synaptic complex, the assembly of which can be driven by DNA conformation changes or by the binding of accessory proteins. Another interesting feature of these reactions is the fate of the donor DNA site, once the intervening sequence has been excised (outlined in [6,18]). In “conservative” site-specific excision systems, which involve the transient formation of a covalent DNA-recombinase intermediate, the flanking arms of the donor site are joined in the final step of the reaction, and the excised sequence is released as a covalently closed circle. In contrast, the excision of cut-and-paste transposable elements or the V(D)J recombination of immunoglobulin genes in E-mail address: [email protected] (M. Bétermier). 0923-2508/$ – see front matter  2004 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2004.01.017

vertebrates leave double-strand breaks at the donor sites, which are processed and healed by cellular DNA repair systems (end joining or homologous recombination). During the formation of the somatic genome of ciliates, which constitute a group of unicellular eukaryotes, largescale DNA elimination takes place in a developmentally programmed manner. In contrast to previously described DNA deletion systems, this process affects numerous loci throughout the whole genome and can be induced experimentally during the sexual events that follow cell starvation (reviewed in [21,42,52]). Furthermore, recent advances have suggested that the germ line sequences that are targeted for deletion are not recognized simply through classical sequence-specific DNA–protein interactions. To illustrate this idea, this review will focus on the current knowledge of DNA elimination and of its regulation in one group of species, Paramecium aurelia.

2. Macronuclear development and DNA elimination in Paramecium 2.1. Nuclear dimorphism and macronuclear development A hallmark of ciliates is the separation of the somatic and germ line genomes into two different nuclei, the macronucleus and the micronucleus, which coexist within the same cytoplasm. The polyploid macronucleus, also called the so-


M. Bétermier / Research in Microbiology 155 (2004) 399–408

Fig. 1. Schematic representation of macronuclear development in Paramecium. In the vegetative parental cell (generation n), the germ line micronuclei are drawn as white circles and the somatic macronucleus is shown in black. Sexual processes are initiated by micronuclear meiosis, which generates eight haploid nuclei, one of which divides once to produce two identical haploid gametic nuclei (not shown in the figure). In the meantime, the macronucleus becomes fragmented. During the fertilization step, fusion of two gametic nuclei gives a diploid zygotic nucleus (see [16] for a detailed description of these early nuclear events). The zygotic nucleus divides twice to produce four identical diploid nuclei: two of those will become the new micronuclei (white circles) and the other two will differentiate into the new developing macronuclei, or anlagen (shown in gray). From this stage, macronuclear development extends over two cell cycles. During the first cycle, intense DNA replication and massive genome rearrangements take place within the anlagen. At the first cell division (or karyonidal division), the two developing macronuclei become separated into each daughter cell. Active DNA synthesis during the second cell cycle accounts for the final ploidy levels reached in the mature macronuclei. Macronuclear development is completed at the end of the second cell cycle and vegetative growth resumes (generation n + 1). The fragments of the parental macronucleus (black dots) are diluted out in the course of the subsequent cell divisions.

matic nucleus, divides amitotically during vegetative growth and is actively transcribed, thus controlling the cell phenotype. It is lost, however, during sexual processes and does not transmit its genome to the progeny. The diploid micronucleus, which divides by mitosis, is transcriptionally silent during vegetative growth, but undergoes meiosis at each sexual cycle and transmits the germ line genome to the zygotic nucleus issued from fertilization. In Paramecium, two successive divisions of the zygotic nucleus produce four identical diploid nuclei, all carrying the germ line version of the genome. Two of those become the new micronuclei, while the other two differentiate into developing new macronuclei, or anlagen. The whole process of macronuclear development extends over two cell cycles following the formation of the zygotic nucleus (Fig. 1): at the first cell division (or karyonidal division), one anlage is distributed to each daughter cell, and mature macronuclei with a final ploidy of 800– 1000 n are obtained after the second cell fission. 2.2. Developmentally programmed elimination of germ line sequences can be precise or imprecise In addition to genome amplification, a comparison of the respective genomic content of the micro- and macronucleus indicates that, within the anlagen, extensive and programmed DNA rearrangements participate in the formation of the macronuclear somatic genome, in a highly reproducible manner from one sexual generation to the next. A significant portion of germ line sequences is eliminated from the developing macronuclear genome, which represents between 10–95% of the micronuclear genome according to ciliate species but has not been quantified precisely in

Fig. 2. DNA elimination during the formation of the macronuclear genome in Paramecium. Germ line regions that are eliminated imprecisely in association with chromosome fragmentation, are shown as a hatched box in the micronuclear genome (top), and precisely excised IESs are drawn as black boxes between two TA dinucleotides. The white arrow represents an open reading frame. Only a few corresponding somatic chromosomes are shown at the bottom of the figure, and the two alternative products of imprecise elimination of repeated germ line sequences (chromosome fragmentation or variable internal deletions) are represented. At the ends of macronuclear chromosomes, telomeric repeats are drawn as gray boxes and their heterogeneous addition points are boxed.

Paramecium [42]. In this ciliate, two types of DNA elimination events have been described (Fig. 2). Imprecise elimination of repetitive germ line sequences (i.e., transposons or minisatellites) has recently been suggested to result either in the fragmentation of germ line chromosomes into shorter acentromeric macronuclear “chromosomes”, carrying variable ends to which telomeric repeats are added, or in the

M. Bétermier / Research in Microbiology 155 (2004) 399–408


imprecise rejoining of flanking sequences [30]. In the latter case, heterogeneous internal deletions can reach a few kbp in size, and occur between variable short direct repeats containing at least one 5 -TA-3 dinucleotide. The second type of eliminated sequences, the short internal eliminated sequences or IESs, have been reported to interrupt open reading frames but can also be found in non-coding regions, including introns (see [16] for a review). Based on an extrapolation of the available sequencing data, Paramecium IESs have been estimated to be around 50 000–60 000 per haploid genome, each one being present as a single copy. The elimination of IESs is an efficient and precise DNA excision reaction (see Section 3.2) that occurs between two defined TA dinucleotides, one copy of which remains at the chromosomal donor site after excision of the intervening sequence [3,17]. Given the large number of IESs found in open reading frames, such a precision at the nucleotide level is required for the reconstitution of a functional somatic genome. 2.3. Timing of macronuclear development Time-course analyses of synchronized P. tetraurelia cells have provided some insight into the chronology of events that take place within the developing anlagen (Fig. 3). Pulselabelling experiments have revealed synchronous and discontinuous peaks of DNA synthesis during the first cell cycle, while a switch to a more continuous mode of DNA amplification is observed following karyonidal division [2]. In the meantime, the parental macronucleus gets fragmented and the resulting fragments, which rapidly stop to replicate, persist inside the cytoplasm and contribute to about 80% of total RNA synthesis throughout the whole period of macronuclear development. Semi-quantitative PCR and Southern blot analysis of the DNA content of the anlagen have indicated that most DNA rearrangements take place during the first cell cycle following zygotic nucleus formation ([3,17] and O. Garnier and A Le Mouël, personal communication). By the time karyonidal division is completed, essentially all germ-line-specific sequences have been eliminated—either precisely or imprecisely—from the genome of the developing somatic macronucleus (Fig. 3). Thus, as was reported for other ciliates [1,13,14,41,49], replication, transcription and DNA rearrangements occur concomitantly in the developing macronucleus. However, in Paramecium, a functional relationship between these events has not been clearly established.

3. Precise IES excision in Paramecium: a two-step mechanism involving specific DNA double-strand breaks 3.1. Paramecium IESs belong to the family of “TA” IESs All of the ∼80 Paramecium IESs that have been identified and sequenced so far (reviewed in [16]) belong to

Fig. 3. Chronology of molecular events within the developing macronucleus of Paramecium. Only the first cell cycle is shown in detail, since all DNA rearrangements take place during this period. Because macronuclear development lasts for a variable number of hours according to growth conditions (20–22 h in rich medium, more than 50 h in starved medium), the time-scale is represented as fractions of the first cell cycle. Peaks of DNA synthesis are adapted from [2]. Global transcription in the anlagen starts early during macronuclear development and can be detected by the first quarter of the first cell cycle. It increases gradually from 6% of total cellular RNA synthesis during the first cell cycle to 80% at the end of the second cycle [2]. The timing of IES excision and circle accumulation was reported in [3,16]. IESs are massively and precisely excised halfway through the first cell cycle, after several rounds of genome amplification have taken place; the earliest excision events can be detected as early as around the first third of this cycle [16,17]. A detailed time-course analysis of the imprecise deletion events associated with chromosome fragmentation is still lacking, but imprecisely eliminated sequences appear to be degraded actively during karyonidal division (O. Garnier and A. Le Mouël, personal communication), together with the most abundant circular excision products generated for IESs longer than 200 bp [16]. These observations suggest that a common degradation pathway may exist for all Paramecium germ line sequences that are being removed from the developing somatic genome.

the family of “TA” IESs, also found in other ciliate species such as Euplotes crassus [19]. A feature common to all Paramecium IESs is their high A/T rich content (80% compared to 68% for macronucleus-destined regions) and their apparent lack of coding capacity. These sequences range from 26 to 882 bp in size, 75% being shorter than 100 bp. IESs are each thought to be unique in the haploid germ line genome, but a degenerate, although statistically significant, consensus sequence (5 -TAYAGYNR-3 ) has been derived from the analysis of 20 IESs, and defines terminal inverted repeats at their ends ([25] and Fig. 4A). This 8-bp sequence includes the TA dinucleotide conserved at IES boundaries, and is very similar to the ends of Tc1/mariner transposons, which duplicate a target TA dinucleotide upon insertion. This similarity was extended to the ends of “TA” IESs of E. crassus, from the genome of which multicopy


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(B) Fig. 4. Structure and sequence analysis of Paramecium IESs. (A) IES structure and general consensus for the ends. The IES is drawn as a black box flanked by two TA repeats and white triangles indicate the additional six nucleotides that define the consensus for IES terminal inverted repeats [25]. The consensus for Paramecium IES ends is aligned with the terminal inverted repeats of Tennessee-related transposons of P. primaurelia [30] and with those of Tc1/mariner elements. The consensus for E. crassus short IESs and Tec transposon-like elements is displayed at the bottom of the figure. Y = C or T, R = A or G, K = G or T, S = C or G (adapted from [19]). (B) Statistical analysis of the sequence of IES boundaries. This study was based on the available sequences of 78 IESs and of their flanking DNA, from the micronuclear versions of the genes encoding the surface antigens A51 , B51 , α-51D, ε-51D and G51 , and of the ICL1d, PAK1, PAK11 and pwB genes of P. tetraurelia strain 51, the A29 and sm19 genes of P. tetraurelia strain d4.2, the 156G and 156ψG loci and one chromosome fragmentation region of P. primaurelia strain 156 (see [16] for references). Special care was taken to take into account the nucleotide composition bias observed in the Paramecium genome. For each position, labelled relatively to the conserved TA, the observed occurrence number of a given nucleotide was normalized by the overall frequency of this nucleotide in the whole panel of sequences (independent frequencies were calculated for macronucleus-destined flanking sequences and for IESs). At each position, normalized occurrence numbers were used for the calculation of the compensated percentage of occurrence of each nucleotide: with this method, a compensated percentage of 25% reflects the random occurrence of one nucleotide.

Tc/mariner-like transposons, the Tec elements, are massively eliminated during macronuclear development [20,22]. It has therefore been proposed that “TA” IESs have evolved from ancient transposons present in the germ line genome by losing their coding capacity, while being kept under selective pressure for their precise excision [26]. The identification, in the germ line genome of P. primaurelia and P. tetraurelia, of transposon-like sequences sharing similar consensus sequences at their ends supports this hypothesis ([30] and O. Garnier and A. Le Mouël, personal communication). No evidence has been obtained thus far, however, for the precise developmental excision of Paramecium transposons, which, rather, appear to be associated with imprecisely deleted regions (see Section 2.2). Another non-exclusive explanation for the origin of Paramecium IESs has been based on the comparison of allelic IESs and proposes that the need to eliminate a given germ-line-limited sequence may impose strong constraints on the adaptive convergent evolution of the ends of the deleted DNA segment, to give a better match to the consensus [8]. Ciliates appear to have evolved a drastic way of inactivating transposons and related sequences by removing them from their somatic genome [30,51,53]. The puzzling observation that IESs and imprecisely eliminated sequences have been maintained in the germ line genome of Paramecium, and of ciliates in general, has raised interesting questions about the possible biological functions of these sequences in the micronucleus. They could play a structural role in mitosis and meiosis, as both of these functions are restricted to the micronucleus and are not supported by the macronucleus (discussed in [52]). In addition, programmed elimination of these sequences can provide a means of fine-tuning gene expression during macronuclear development, as was demonstrated for the gene encoding the de novo telomerase catalytic subunit of E. crassus [24]. Because it is interrupted by an IES, this gene can only be expressed after excision has taken place. Furthermore, it is located within a region that is eliminated during the process of chromosome fragmentation: this ensures that the de novo telomerase gene is switched off once the new chromosome ends have been formed. 3.2. IES excision involves site-specific double-strand breaks centered on the conserved TA at each end While the molecular mechanisms that underlie the imprecise deletion of germ line repeated sequences still remain essentially unknown in Paramecium, recent progress has been made towards the understanding of the reaction that leads to the precise elimination of IESs. During the first cell cycle of macronuclear development, extrachromosomal molecules are detected for IESs longer than 200 bp [3,16]. These excised IESs accumulate mostly as doublestranded covalently closed circular molecules that are degraded by an active mechanism before karyonidal division is completed. In these circles, IES ends are precisely joined

M. Bétermier / Research in Microbiology 155 (2004) 399–408

Fig. 5. Model for IES excision in Paramecium. The proposed mechanism involves the introduction of 4-base staggered double-strand cuts, one at each IES end, and subsequent DSB repair (adapted from [17]). The 5 nucleotide of each 4-base overhang is indicated by a circle, the recessive 3 -end by an arrowhead. After double-strand cleavage, broken chromosome ends (thin lines) align within a junction repair intermediate (step 1), and the macronuclear junction results from the processing of the 5 flapped nucleotides (step 2), fill-in of the 3 recessed ends (dotted lines in step 3) and ligation. The ends of the putative linear excised IES (thick lines) may be joined within a similar intramolecular intermediate that will be processed to give the circles.

by one copy of the flanking TA repeat. This distinguishes Paramecium IESs from those of E. crassus, which exhibit more heterogeneous circular junctions, with two copies of the TA dinucleotide separated by a 6/10-bp heteroduplex region originating from the flanking macronucleus-destined sequences [27]. Although IES circles may be only secondary products of the reaction, the detection of these extrachromosomal molecules and the precision in the formation of chromosomal and circular junctions have provided evidence that Paramecium IESs are removed from the somatic genome through a site-specific excision reaction, involving DNA cuts in the vicinity of each flanking TA. The current model for IES excision in Paramecium is based on the mapping of the free 3 OH and 5 PO4 groups released at IES boundaries during the formation of a new macronucleus, and accounts for the observed fidelity of chromosome junction formation [17]. In an initiating step, two 4-base staggered double-strand cuts, one at each end, are introduced on each side of the conserved TA dinucleotides that define IES boundaries (Fig. 5). This generates two broken chromosome ends at each excision site, with 4-base 5 overhangs carrying a central TA. The second step of the reaction allows precise and efficient closure of the chromosomal junction. Alignment of the two 5 protruding chromosome ends is thought to be mediated through the


pairing of the TA dinucleotides carried by each singlestranded extension. Chromosome closure would then require controlled processing of the aligned ends, including removal of the 5 terminal residue, which is generally mispaired within the end alignment intermediate, and gap filling from their recessed 3 -end, prior to the final ligation step. In this model, excised IESs are expected to be released as linear molecules carrying similar 4-base 5 overhangs at their ends, which, according to their length or flexibility, could in turn be circularized in an intramolecular end-joining reaction. A similar two-step model, involving the doublestrand staggered cleavage of both IES ends, followed by the alignment of broken ends and the repair of chromosome and circular IES junctions, has been proposed for the excision of E. crassus “TA” IESs [22,27], although DNA cleavage sites have not been mapped in this ciliate. These models differ radically from those previously proposed for IES excision in other ciliate species. In Tetrahymena, doublestrand breaks were also detected at the ends of IESs, but excision in this ciliate is thought to be initiated by the cleavage of one end only, followed by the nucleophilic attack of the other end to generate directly the macronuclear junction on one strand [45,50]. A related model involving a single initiating cut was also proposed for IES excision in Oxytricha [50]. Important points are to be addressed before a complete picture of the excision reaction in Paramecium can be drawn. In particular, whether both IES ends are recognized and cleaved independently or in a concerted manner remains a fully open question. The partners that participate in DNA cleavage and subsequent joining of chromosome ends will also have to be identified. As previously discussed [16,17], possible DNA cutters could be cellular transposase-like enzymes or other endonucleases related to site-specific recombinases or topoisomerases. More work is clearly needed to identify the machinery involved, since no firm conclusion can be drawn from the currently available data on the structure of IES excision intermediates. Finally, the two modes of DNA elimination described in Paramecium—precise excision of unique “TA” IESs and imprecise elimination of micronucleus-specific repeated sequences—share one striking common feature [30]. Both types of reactions occur between short direct repeats, one of which is maintained at the chromosomal junction in the macronucleus, and in both cases the repeats contain at least one TA dinucleotide. Even though no experimental evidence has been obtained for the cleavage of imprecisely eliminated sequences, this raises the interesting possibility that the same DNA cutting machinery may be involved in both deletion events. Additional mechanisms, such as the precise targeting of specific TA dinucleotides or the protection of cleaved DNA against exonucleolytic degradation, would ensure the precise excision of IESs.


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4. Targeting Paramecium germ line sequences for deletion 4.1. Determination of DNA cleavage sites: more than the specific recognition of a DNA sequence motif Precise mapping of the double-strand breaks generated at each IES end during excision has suggested that the site for initial cleavage is located 1 bp into the macronucleusdestined sequences that flank the conserved TA (Fig. 5 and [17]). In agreement with this observation, the analysis of 78 IES ends reveals a slight, but significant, sequence bias at the three positions adjacent to the TA, within the macronucleus-destined sequences (Fig. 4B). This has led to the definition of an enlarged consensus sequence for IES ends, which includes the absolutely conserved TA and its surrounding nucleotides (5 t43 g44 g44 T100 A100 Y93 A47 G73 Y79 Y63 R76 R73 3 , where lower case letters indicate nucleotides maintained in the macronucleus, bold letters the TA dinucleotide marking the IES boundary and upper case letters, the residues inside the IES; see legend to Fig. 4 for details about score calculations). Genetic evidence has confirmed that the presence of TA dinucleotides at both ends is required for excision: a point mutation within the TA at a single boundary results in maintenance of the mutant IES in the macronucleus [34,44]. Mutations at the more variable 8th position of the enlarged consensus sequence also have an inhibitory effect, although low levels of precise excision have been reported in one case [31,33,35]. In addition, deletion experiments have suggested that external macronucleus-destined sequences are also involved in excision [29]. Intriguingly, however, neither a full match to the consensus nor the conservation of terminal inverted repeats, except for the flanking TAs, are required for an individual IES to be excised efficiently. Rather than representing a specific recognition site for DNA binding, the consensus, therefore, may reflect the existence of a preferred sequence context for IES end cleavage. Furthermore, as will be emphasized below, the mere nucleotide sequence of its ends is probably not sufficient to define an IES, since sequences matching the consensus can be found in many non-excised regions of the macronuclear genome of Paramecium [25]. Although this still has to be proven, the overall A/T-richness of IESs might also favor the formation of secondary DNA structures or the opening of the DNA double helix, which could facilitate their recognition by the excision machinery. In addition to the problem of the recognition and cleavage of IES ends, one may wonder how the second class of Paramecium repeated germ line sequences are targeted for deletion, since these exhibit much less precisely defined boundaries, with no other obvious common sequence feature than the presence of one or several TA dinucleotides. Some speculations have been made about a putative role of transcription in the developing macronucleus, or of noncoding transcripts, in triggering the elimination of specific germ line sequences. Indeed, global RNA synthesis, as mon-

itored by the incorporation of tritiated uridine, was detected in P. tetraurelia anlagen prior to the massive excision of IESs (Fig. 3 and [2]), but whether germ-line-limited sequences are transcribed during this period has not been assessed in this ciliate. In contrast, this point was addressed in T. thermophila, in which bi-directional transcription of IESs, some of which is initiated from outside flanking sequences, was detected several hours before their elimination from the new macronucleus [5]. However, whether this early read-through transcription is restricted to the micronucleus or can originate also from the developing macronucleus has not been established unambiguously. Although more work is clearly needed to examine whether specific transcription of developmentally eliminated sequences does occur inside Paramecium anlagen, this may provide a good way of directing the excision machinery to specific loci in the genome. As was evidenced for other site-specific recombination systems, such as the V(D)J rearrangement of immunoglobulin genes (reviewed in [15]), transcription per se may positively regulate the accessibility of recombination sites by inducing the local opening of chromatin through nucleosome remodelling or histone modification. Alternatively, a functional role could be proposed for the resulting transcripts: by pairing to their homologous DNA sequences, they could target DNA cutting enzymes to specific sites (e.g., the boundaries of eliminated sequences). One illustration of this idea is provided by the class switch recombination (CSR) that takes place within the genes encoding the constant regions of immunoglobulins in vertebrates (see [32] for a review): in this system, annealing of the nascent transcript to its template DNA strand generates an R-loop structure, which was shown recently to be a key early intermediate of the CSR reaction [46,55]. These first two models may apply, in Paramecium, to the targeting of all germ-line-restricted sequences, whatever the final precision of their excision may be. Another type of mechanism has been proposed recently in Paramecium and Tetrahymena [30,51], in which putative developmental transcripts produced in the anlagen from repeated germ line sequences would direct the selective deposition of an epigenetic mark, such as the formation of heterochromatin, onto the whole length of their homologous genomic sequences, thereby targeting these sequences for imprecise elimination (see also Section 4.2.2). 4.2. Trans-nuclear regulation of germ line sequence elimination: a homology-dependent epigenetic mechanism While addressing the issue of target choice specificity during DNA rearrangements in ciliates, the anlagen should not be considered as isolated entities in the cell. As illustrated for Paramecium in Fig. 6, each new macronucleus develops in the presence of the micronuclei and of the parental macronucleus, which, while undergoing progressive degradation, is still present and transcribed throughout macronuclear development. The existence of an additional level of regulation of DNA rearrangements stems from this unique

M. Bétermier / Research in Microbiology 155 (2004) 399–408

Fig. 6. DAPI-staining of a P. tetraurelia cell undergoing macronuclear development, prior to karyonidal division (adapted from [3]). Arrows point to the two developing macronuclei, or anlagen. The surrounding stained structures are the fragments of the parental macronucleus. Micronuclei do not show up in this picture.

property of ciliates, which harbor different types of nuclei coexisting in a unique cytoplasm. Recent data indicate that ciliates have evolved a sophisticated system to compare, on a genome-wide scale, the DNA content of their different nuclei and use this comparison to eliminate all germ-linerestricted sequences. Evidence for a trans-nuclear control of both precise and imprecise developmental deletion events was provided by the discovery that alternative rearrangement patterns may be inherited in a non-Mendelian manner in Paramecium and Tetrahymena (reviewed in [38,39]). The excision of a subfraction of Paramecium IESs is, indeed, controlled by the genomic content of the parental macronucleus: injection of a DNA fragment carrying one IES into the macronucleus of a cell can inhibit the precise excision of the homologous sequence in the newly formed macronucleus of the following sexual generation (Fig. 7A). This may even result in the establishment of stable variant cell lines harboring a fully wild-type germ line genome, but in which a given IES is no longer excised from the macronuclear genome [9,10]. This effect is strictly homology-dependent, since all other IESs are excised normally. IES excision in Tetrahymena was shown to be submitted to the same kind of epigenetic control [4], although the boundaries of deleted elements are less precisely defined in this ciliate and, in particular, do not contain any conserved TA dinucleotide. With respect to imprecise deletion of germ line sequences in Paramecium (Fig. 7B), another variant cell line has been described, in which an alternative chromosome fragmentation site is used reproducibly from one sexual generation to the next, which results in the deletion of a locus normally maintained in the macronucleus, even though the germ line genome is wild-type [12]. Strikingly, transformation of the deleted parental macronucleus with a DNA fragment carrying a portion of the germ line sequence homologous to the deleted



(B) Fig. 7. Epigenetic control of developmentally programmed DNA elimination in Paramecium by the parental macronucleus (adapted from [39]). (A) Control of IES excision. In the wild-type cell line, all IESs (black boxes) present in the micronuclear genome are precisely excised from the genome of the developing macronucleus of the next sexual generation. Transformation of the macronucleus with a DNA fragment carrying one IES can block the excision of the homologous IES from the macronuclear genome of the next sexual generation: this may result in the establishment of an IES+ cell line, harboring a wild-type germ line genome but in which one IES is never excised. (B) Control of imprecise elimination associated with chromosome fragmentation. The d48 cell line carries a wild-type micronuclear genome, but exhibits a variant pattern of chromosome fragmentation relative to the wild-type cell line. This corresponds to imprecise elimination of the surface antigen A gene (represented by a hatched box). If the macronucleus of the d48 mutant is transformed with a fragment of the A gene, restoration of the wild-type pattern of chromosome fragmentation can be observed in the next sexual generation (rescued cell line).

locus can restore a wild-type fragmentation pattern, i.e., the amplification of the homologous region, in the macronucleus of the next sexual generation ([23,28,54] and Garnier et al., submitted).


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Fig. 8. Models for the genome-wide comparison of the somatic and germ line versions of the genome in ciliates. The left side of each panel shows a Paramecium cell undergoing macronuclear development: the dotted-lined arrow represents the export of RNA molecules from the micronuclei (white circles) to the parental macronucleus (black dots), while RNA transport from the parental macronucleus to the anlagen (gray circles) is symbolized by a full-lined arrow. An enlarged representation of the developing macronucleus is drawn on the right of each panel. (A) scnRNAs (26–31 bp), processed from micronuclear transcripts (1) and subtracted for sequences homologous to those present in the parental macronucleus (2), would induce the epigenetic modification of the chromatin associated with homologous germ line-restricted sequences in the developing macronucleus and target them for deletion [40]. (B) Non-coding RNAs produced by the parental macronucleus would protect homologous sequences of the anlagen against deletion (Garnier et al., submitted). (C) In a variation of the template-guided recombination model [43], RNA molecules imported from the parental macronucleus would pair to homologous sequences in the genome of the developing macronucleus and guide the correct formation of chromosomal excision junctions.

Taken together, these observations have led to the suggestion that a trans-nuclear cross-talk between the parental macronucleus and the anlagen is involved in the determination of the fate of a germ-line sequence—deletion or amplification—in the developing macronucleus. Because these phenomena depend on sequence homology, the emerging idea has been that this information exchange is mediated through the pairing of nucleic acids originating from either type of nuclei. As a consequence of this large-scale genome comparison, germ line sequences absent from the parental somatic genome would be eliminated from the new developing macronucleus. In a first model, Mochizuki et al. [40] have proposed that germ line sequences are selected and targeted for elimination through the sequence homologydependent pairing of short non-coding RNAs (Fig. 8A). Based on experimental data obtained in Tetrahymena [5], they have suggested that early bi-directional transcription of all germ line sequences takes place in the micronucleus during the early steps of macronuclear development. The result-

ing double-stranded transcripts would be exported through the cytoplasm to the parental macronucleus and would get processed into short double-stranded (ds)RNAs, or scnRNAs, by an enzymatic activity related to the RNAi pathway. These 26- to 31-bp scnRNAs would then be compared to the DNA content of the parental macronucleus, and all molecules matching macronuclear genomic sequences would get degraded. Finally, only scnRNAs specific for germ-linelimited sequences would escape this selection and be transported to the developing anlagen, where they would recognize homologous DNA sequences and guide the epigenetic modification of their associated chromatin. A DNA cutting machinery would introduce DNA breaks at the boundaries of the modified region and clip out the intervening germ line sequences. The idea of an RNA-guided selection of eliminated sequences was bolstered recently by the demonstration that injection of a long dsRNA into the cytoplasm of conjugating Tetrahymena cells triggers the developmental, imprecise deletion of the homologous DNA sequence from the developing macronucleus [53]. It has been reported, furthermore, that heterochromatin formation is associated with the developmental elimination of germ line sequences in Tetrahymena [11,48]. However, while such a heterochromatin-driven deletion model would nicely explain imprecise deletion events, precise excision reactions probably require some additional component to specify the deletion boundaries. In addition, the extremely short size of Paramecium IESs, which can be as short as 26 bp, is difficult to reconcile with the assembly of a specialized chromatin structure, since a nucleosome usually covers around 140 bp. Other authors have adopted an opposite point of view and proposed an alternative model, in which elimination would be the default fate of all sequences in the developing macronucleus (Garnier et al., submitted for publication). In this model, developmental signals (e.g., long non-coding transcripts) would be produced by the parental macronucleus rather than by the micronucleus, then would be exported to the anlagen (Fig. 8B). These molecules would mark homologous sequences in the developing new macronucleus and protect them against elimination: germline-restricted sequences would therefore be excluded during developmentally programmed DNA rearrangements. A related “template-guided recombination” model, involving a homology-dependent alignment of the macro- and micronuclear versions of the genome, could further account for the precise positioning of IES ends ([43] and Fig. 8C). In Paramecium, all trans nuclear effects described in Fig. 7 can be explained by any of these models, although no direct experimental evidence has been obtained in this ciliate to support the existence of developmental non-coding transcripts that may regulate DNA rearrangements. One additional observation is harder to reconcile directly with the scnRNA model for the targeting of chromatin modifications to eliminated germ line sequences. It has been reported, indeed, that injection into the macronucleus of vegetative cells of high copy numbers of a DNA fragment homologous to a

M. Bétermier / Research in Microbiology 155 (2004) 399–408

sequence normally present in the macronuclear genome can induce the imprecise deletion of the homologous region in the developing macronucleus of the next sexual generation [36,37]. In this case, however, all micronuclear transcripts homologous to the injected transgene should be subtracted in the transformed parental macronucleus, and there should be no scnRNAs produced to label the homologous sequences in the developing macronucleus as germ-line-limited and target them for elimination. Interestingly, it has been shown that short dsRNAs produced from the injected DNA fragment do accumulate in vegetative cells and throughout the development of the macronucleus of the next sexual generation (Garnier et al., submitted). In addition, the same imprecise deletions can be recovered if P. tetraurelia cells are fed on bacteria producing dsRNAs homologous to the target sequence. In Paramecium also, therefore, short dsRNAs can induce the deletion of homologous sequences in the developing macronucleus. Their action could be direct, through the sequence-specific formation of a heterochromatin structure at each deleted loci, or indirect, through the triggering of an RNAi-like pathway for the degradation of a positive RNA signal that would otherwise protect macronucleus-destined sequences against deletion.

5. Concluding remarks Our current knowledge of developmentally programmed genome rearrangements in Paramecium points to the existence of two different modes of DNA elimination in this ciliate. One potentially interesting direction for future research will be the identification of the common and distinctive features of these two processes. The deletion of short, unique IESs is achieved through a site-specific excision reaction that involves the double-strand cleavage of both IES ends. It is also characterized by a striking fidelity in the repair of the broken excision donor site. How this precision is reached remains elusive, especially in the absence of any information on the proteins and other partners involved in the reaction. Even less experimental data is available regarding the molecular steps that lead to the imprecise deletion of repeated germ line sequences, and it is presently not known whether the same enzymatic machineries participate in both reactions. Finally, perhaps the most challenging issue for the coming years will be the elucidation of the molecular basis of the homology-dependent epigenetic control of DNA rearrangements in Paramecium and other ciliates. Identification of the development-specific non-coding RNAs involved in this regulation and of the mechanism(s) of their action will certainly enlarge our current view of how site-specific recombination machineries may recognize their target sites in a genome. This may also provide a conceptual framework for the understanding of how some characters, such as the mating type of Paramecium (reviewed in [47]), can be maternally inherited through the cytoplasm from one sexual generation to the next.


Acknowledgements I would like to thank Ariane Gratias and Gersende Lepère for their fruitful collaboration and all other members of Eric Meyer’s group for extremely rich and stimulating discussions. Special thanks also to Sandra Duharcourt and Eric Meyer for their comments on the manuscript. Work in our laboratory has been supported by the Centre National de la Recherche Scientifique, the Comité de Paris de la Ligue Nationale contre le Cancer (grant No. 75/01-RS/73) and the French Ministry of Research (Programme de Microbiologie: Microbiologie fondamentale et appliquée, maladies infectieuses, environnement et bioterrorisme).

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