Artificial and Epigenetic Regulation of the I Factor ... - Semantic Scholar

Copyright  2000 by the Genetics Society of America

Artificial and Epigenetic Regulation of the I Factor, a Nonviral Retrotransposon of Drosophila melanogaster Emmanuel Gauthier,*,1,2 Christophe Tatout†,1 and Hubert Pinon* *Centre de Geńe´tique Molećulaire et Cellulaire, CNRS UMR 5534, Universite´ Claude Bernard, F-69622 Villeurbanne Cedex, France and †Biogemma, F-63177 Aubie`re Cedex, France Manuscript received February 3, 1999 Accepted for publication August 28, 2000 ABSTRACT The I factor (IF ) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.

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RANSPOSABLE elements are mobile DNA sequences found in all organisms from bacteria to higher eukaryotes. In eukaryotes, transposable elements represent a substantial fraction of the genome: at least 15% of the Drosophila genome, 35% of the human genome, and more than 50% of some plant genomes (Finnegan 1992; San Miguel et al. 1996; Voytas and Naylor 1998). Their mobility is associated with deleterious effects such as insertional mutagenesis and chromosomal rearrangements (Lim and Simmons 1994), but it is also believed to contribute positively to the evolution of the host genome (reviewed in Kidwell and Lisch 1997). Transpositions are usually rare events and therefore are difficult to study, except during the hybrid dysgenesis process (Bre´gliano and Kidwell 1983). Hybrid dysgenesis is observed in Drosophila melanogaster when flies containing silenced but potentially active transposable elements such as P, the I factor (IF), or hobo are crossed with a strain devoid of autonomous

Corresponding author: Hubert Pinon, Centre de Geńe´tique Molećulaire et Cellulaire, UMR 5534 baˆt. 741, Universite´ Claude Bernard, 43, Blvd. du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France. E-mail: [email protected] 1 These authors contributed equally to this work. 2 Present address: Institut Gustave Roussy, CNRS UMR 1573, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France. Genetics 156: 1867–1878 (December 2000)

elements (Rubin et al. 1982; Fawcett et al. 1986; Blackmann et al. 1987). In the germline of the progeny the elements are mobilized at high frequency, and sterility is observed. For that reason hybrid dysgenesis has been proposed as one of the mechanisms that contribute to the isolation of subpopulations and that are involved in speciation (Bre´gliano and Kidwell 1983; Kidwell and Lisch 1997; O’Neill et al. 1998). In the case of the IF, a high level of transposition occurs only when IFcontaining males (from inducer, or I strains) are crossed with IF-lacking females (from reactive, or R strains). IF, which is silenced in I strains, is subsequently mobilized at high frequency in the germlines of the female progeny, which exhibit the specific traits of I-R hybrid dysgenesis, the most conspicious one being a more or less severe sterile female (SF) phenotype. These so-called SF females lay a normal number of eggs, but the eggs do not hatch because of early embryonic death (Lavige 1986). The reciprocal cross (I female ⫻ R male) gives rise to RSF females, which show normal fertility and a fivefold lower transposition rate than SF females (Picard 1978). In ovaries from SF females IF silencing is disrupted and IF comes under the positive control of a quantitative trait, called reactivity, which is provided by the R strain and shows a complex mixture of chromosomal and maternal inheritance (reviewed in Bre´gliano and Kidwell 1983; Finnegan 1989); reactivity is for the most part maternally inherited from one generation to the

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next. However, the genetic determinants appear to be chromosomal (Picard 1978; Bucheton and Picard 1978). The reactivity level of an R female is measured by the percentage of sterility of its SF daughters. Accordingly, it is possible to identify strong and weak reactive strains (Bucheton et al. 1976). Reactivity level can be reversibly modified over several generations by various experimental conditions: female aging, breeding temperature (Bucheton 1978, 1979a,b; reviewed in Bre´gliano and Kidwell 1983; Finnegan 1989), inhibitors of DNA synthesis, and gamma rays (Bre´gliano et al. 1995). In addition, the reactivity level controls the transcriptional activity of the IF (Lachaume and Pinon 1993; McLean et al. 1993; De La Roche Saint Andre´ and Bre´gliano 1998), the cellular localization of the IF translation products (Seleme et al. 1999), and the percentage of crossing over in the maternal germline (Laurençon et al. 1997). Reactivity is also believed to exist in I strains although it induces neither sterility nor high rates of IF transposition, which suggests that in I females IF autoregulation interferes with reactivity (Laurençon et al. 1997). Reactivity has been likened to an error-prone recombinationrepair process called the variability modulation system (VAMOS), thought to resemble the SOS system in bacteria (Bre´gliano et al. 1995; Laurençon et al. 1997). Indeed, the SOS system shares two interesting similarities with reactivity: it is able to regulate the expression of the transposable element Tn5 through the LexA protein (Echols and Goodman 1990), and it leads to insertional mutagenesis when induced (Kuan and Tessman 1991). However, whether IF transposition is directly responsible for SF sterility or whether it leads to the overinduction of the VAMOS system has yet to be resolved. In the generations following a dysgenic cross, the IF invades the genome until it reaches 10–15 copies per haploid genome and is progressively repressed through an autoregulation process (Pe´lisson and Bre´gliano 1987). So far, IF autoregulation has been explained by four nonexclusive mechanisms: (1) The protein encoded by ORF1 of IF contains zinc-finger domains and may be an IF self-repressor. (2) Alternatively, the socalled “titration” model proposed that ovarian-specific activators able to bind and activate the IF promoter are present in a limited amount (Udomkit et al. 1996). As IF copy number increases, the local concentration of this factor on each copy of the IF decreases. This would decrease the overall level of transcription of the IF under a threshold where transposition does not occur. From this hypothesis, Chaboissier et al. (1998) demonstrated that, indeed, the IF promoter induced an increasing repressive effect as its copy number increased. However, deletion of the binding site of the ovarian activator previously described by Udomkit et al. (1996) did not abolish this repression. (3) These data were then reinterpreted (Chaboissier et al. 1998) as indicating a phenomenon analogous to a transcriptional silencing, per-

haps due to a change in IF chromatin structure as described in Drosophila (Pal-Bhadra et al. 1997, 1999). (4) Recently, Jensen et al. (1999a,b) showed that IF autoregulation could be assigned not to the ORF1 polypeptide but to the production of IF RNAs. These RNAs appeared responsible for a phenomenon similar to the post-transcriptional silencing previously described in plants and fungi (Matzke and Matzke 1995; Cogoni et al. 1996; Grant 1999; Selker 1999) and known as cosuppression. Thus, Chaboissier et al. (1998) and Jensen et al. (1999b) agree that IF regulation is controlled through epigenetic phenomena showing the characteristics of homology-dependent gene silencing. IF belongs to the LINE (long interspersed nucleotidic element) family of transposable elements and transposes through reverse transcription of an RNA intermediate ( Jensen and Heidmann 1991; Pe´lisson et al. 1991). Consequently, reducing the level of this RNA is expected to reduce IF transposition. To test this idea, the RNA intermediate of IF has been targeted by artificial antisense RNAs (asRNAs) previously evaluated in transient expression assays (Tatout et al. 1998). Here the use of these transgenic asRNAs demonstrates that downregulation of IF abolishes the sterile female phenotype. Moreover, we show that some transgenes confer a repressive effect with zygotic but also with particular maternal or paternal components. These inherited effects may be induced at the DNA and/or RNA (sense or antisense) level, provided that these sequences contain specific features of the IF. These results support the idea that IF expression is controlled through epigenetic mechanisms, provide a genetic description of this epigenetic phenomenon, and show that the mechanisms involved are reminiscent of reactivity itself.

MATERIALS AND METHODS D. melanogaster strains: Because breeding conditions strongly influence the reactivity level and, consequently, the intensity of the hybrid dysgenesis syndrome, experimental cultures must be grown under carefully controlled procedures. First, strains were reared at 20 ⫾ 1⬚ under uncrowded conditions on the axenic medium of David (1959). Second, all stocks were maintained with short generations; i.e., each generation was obtained from very young (2-day-old) parents. Standard strains used in this study are the R strain wK (Lu¨ning 1981) and the I strain w1118 (Hazelrigg et al. 1984). The R strains LH23 and Valence, which carry balancer chromosomes, are from the stocks of the former Laboratoire de Geńe´tique, Universite´ de Clermont-Ferrand; their I derivatives were obtained by chromosome contamination with I factor sequences after crossing with the standard w1118 strain. Additional information about the genetic markers can be found in Lindsley and Zimm (1992). DNA constructs: Fragments of the IF ORF1 were derived from the pSPI1 clone (Chaboissier et al. 1990). The IF-lacZ fusion is described in Lachaume et al. (1992) and the RZI construct in Tatout et al. (1998). RZAP was derived from RZI using primers P1 to P6 (see below) in recombinant PCR. P1 and P2 contain a BamHI site (underlined) used for cloning;

Regulation of I Factor Transposition P1 allows deletion of the first five nucleotides of IF essential for IF promoter activity (McLean et al. 1993). P3 and P4 mutate ATG187 to TAG (boldface type), giving rise to a new XbaI site (underlined). P5 and P6 change ATG586 to CGT (boldface type), giving rise to a new SnaBI site (underlined). P1 and P4 yield a 198-bp PCR fragment; P3 and P6 yield a 429-bp fragment; P5 and P2 yield a 546-bp fragment. The 429and 546-bp fragments were combined in a subsequent PCR using P3 and P2 to generate a 954-bp fragment. Finally, the 198-bp and verified 954-bp fragments were combined together with P1 and P2 to generate a 1124-bp mutated ORF1 sequence corresponding to RZAP. The BB construct contains the BstBIBstBI fragment from the pSPI1 clone. DNA sequencing was performed using the T7 sequencing kit (Pharmacia, Piscataway, NJ). After appropriate enzymatic treatments, RZI, RZAP, and BB were cloned in antisense orientation in the BamHIdigested pCaSpeR-act5C vector (Thummel et al. 1988), which contains the promoter and the polyadenylation signals from the actin5C gene. The 8S construct is the same as RZI, but this truncated ORF1 fragment was cloned in the pW8 transformation vector (Klemenz et al. 1987) digested by BamHI. P1 ⫽ (⫹6)CGGGATCCACTTCAACCTCCGAAGAGATAAGT CG(⫹33) P2 ⫽ (⫹1104)CGGGATCCATTAGGTGATGGAGTGTTTGT TGTCC(⫹1079) P3 ⫽ (⫹171)CCCTTAACCAACAATCTAGACAGACCCACC (⫹200) P4 ⫽ (⫹197)GGGTCTGTCTAGATTGTTGGTTAAGGGC (⫹170) P5 ⫽ (⫹579)AAAAATACGTAAACGGCAAAACCCC(⫹603) P6 ⫽ (⫹600)GTTTTGCCGTTTACGTATTTTTTTAACTTCA G(⫹569) P-mediated transformation and establishment of transgenic Drosophila stocks: Except that plasmid pUChs⌬2-3 was the source of transposase, P-mediated germline transformation was performed as usual (Rubin and Spradling 1982) in the wK strain that is M and R in the P-M and I-R systems, respectively. The transformation vectors contain the mini-white gene, which allows the selection of transformants on the basis of their colored-eyed phenotype. Inserts were mapped to chromosomes by segregation analysis against balancer chromosomes M5, CyO, and Dcxf; results were confirmed by in situ hybridization on salivary gland polytene chromosomes (Bie´mont 1986). Transgene integrity and copy number were determined by Southern blots probed with I factor or actin5C sequences. For the RZI construct, 10 independent lines bearing one homozygous copy (1RZI lines) were established. Among them, some insertions on the chromosomes X, II, and III were arbitrarily chosen to construct, by standard genetic procedures, three lines with two homozygous insertions (2RZI lines) and two lines with three homozygous insertions (3RZI lines) on the chromosomes II and III, and X, II, and III, respectively. Independent transgenic lines were also obtained for the other constructs: 17 for RZAP (1RZAP lines), 4 for 8S (18S lines) with all insertions on chromosome III, 16 for BB (1BB lines). For the RZAP construct some insertions were also combined to establish three lines with two homozygous insertions on chromosomes II and III (2RZAP lines). For the BB construct, three lines with two (2BB) and two lines with three (3BB) homozygous insertions distributed among the three main chromosomes were established. Ten lines carrying one homozygous copy of RZI have been studied in parallel. In this report, the term 1RZI refers to the “average line”—whatever the chromosomal position of its transgene—and, by extension, depicts the average effects obtained from these 10 single-copy-carrying lines. 2RZI and 3RZI have the same meaning for the lines carrying two or

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three insertions, respectively. In the same way, 1RZAP, 2RZAP, 18S, 1BB, 2BB, and 3BB are used to stand for the respective average line and average effect from all the corresponding transgenic lines. For each construct, all lines were subsequently contaminated by functional I factors (Lachaume and Pinon 1993) to obtain the same set of transgenic lines but in the inducer state. RNA probes and standards: The sense (antisense) RNA probes used in antisense (sense) Northern blot or slot-blot analysis were synthesized from a HindIII- (EcoRI-) digested pSPI1 vector (Chaboissier et al. 1990), using [32P]rUTP and SP6 (T7) RNA polymerase (Boehringer Mannheim, Indianapolis). Sense and antisense RNAs used to calibrate RNA slotblot analysis were obtained in the same way using unlabeled rUTP. Prior to use, sense and antisense RNAs were treated with Dnase I, extracted with phenol-chloroform, and precipitated twice by ethanol. Northern and slot-blot analysis: Ovaries from 5-day-old flies were dissected and stored at ⫺80⬚. Poly(A)⫹ RNAs were prepared using the Quick prep micro mRNA purification kit (Pharmacia). For the 8S transgenic lines, total RNAs were extracted (De La Roche Saint Andre´ and Bre´gliano 1998) because the pW8 transformation vector does not contain a polyadenylation sequence. Extracted RNAs were transferred to nylon N membranes (Amersham, Buckinghamshire, UK) either after electrophoresis in formaldehyde-agarose gels or with a slot-blot apparatus. Before electrophoresis 5 ␮g of poly(A)⫹ RNAs (or 10 ␮g of total RNAs) were denatured for 5 min at 65⬚ in 1⫻ MOPS, 50% formamide, and 7% formaldehyde. For slot-blot analysis, RNAs were denatured 15 min at 68⬚ in 1⫻ SSC, 50% formamide, and 7% formaldehyde. Then, 0.25, 0.5, 1.0, and 1.5 ␮g of poly(A)⫹ RNAs (or 0.5, 1.0, 1.5, and 2.0 ␮g of total RNAs) were loaded on a membrane. In parallel, known quantities of in vitro transcribed antisense or sense RNAs, estimated by UV absorption at OD260 nm, were used as standard. Membranes were hybridized as described in Sambrook et al. (1989). Slot-blots were quantified using the PhosphorImager system (Bio-Rad). For each transgenic line quantifications were performed from three independent extractions to obtain mean values with standard errors. ␤-Galactosidase quantification: Ovarian ␤-galactosidase activities were measured using an ELISA kit (TEBU, 5⬘-3⬘ Inc.). After dissection, 10 ovaries were disrupted in 200 ␮l of PBS containing 1 mm phenylmethylsulfonyl fluoride, frozen in liquid nitrogen and allowed to thaw thrice, and centrifuged 15 min at 15,000 ⫻ g. The supernatant was recovered and checked for ␤-galactosidase activity as described by the manufacturer. Quantifications were performed from at least three independent extractions to obtain mean values with standard errors. Measurement of the sterility level and statistical analysis: Three sets of five to eight SF females from each dysgenic cross were mated with sib males and allowed to lay eggs for 1 wk at 20 ⫾ 1⬚. Every 24 hr, eggs were recovered and allowed to develop for 36 hr. Percentages of hatched and unhatched eggs were scored to evaluate SF sterility. For a given transgene copy number, mean values and standard deviations were estimated from at least three independent experiments performed on all the corresponding strains, unless otherwise stated. For each line and for each dysgenic cross, the SF sterility data were recorded daily during the first 7 days of egg laying and combined to obtain the average sterility level. From these data, zygotic and maternal and paternal effect values were then computed as described in the legend of Figure 3. Finally, data were combined over lines, for each transgene and according to copy number, to give the data presented under the terms 1RZI, 1RZAP, 18S, 1BB, 2RZI, 2RZAP, 2BB, 3RZI, and 3BB (see Table 1 for an example).

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E. Gauthier, C. Tatout and H. Pinon TABLE 1 Quantitative estimations of zygotic, maternal and paternal effects

Constructs RZI

RZAP 8S BB

Zygotic effect

Maternal effect

Copy number

% rescued embryos

LS

1 2 3 1 2 1 1 2 3

35 ⫾ 9 55 ⫾ 5 ND 24 ⫾ 7 42 ⫾ 6 3⫾1 2⫾0 0⫾0 ND

** ** ** ** NS NS NS

Paternal effect

% rescued embryos

LS

% rescued embryos

LS

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

** ** ** ** ** ** NS NS NS

13 ⫾ 6 18 ⫾ 3 ND 12 ⫾ 4 15 ⫾ 6 0⫾0 0⫾1 0⫾0 ND

* **

18 40 88 24 48 24 2 1 0

6 7 2 6 4 5 1 1 0

* * NS NS NS

All effects were computed 10 generations after transgene establishment, as described in Figure 3, A and C. For a given construct and copy number, the data shown are average values from all transgenic lines. Values are expressed as percentage of rescued embryos ⫾SE. ND, not done, one transgenic insertion on the X chromosome being lost by paternal transmission. LS, level of significance for the difference between each value and zero. NS, not significant. *P ⬍ 0.05; **P ⬍ 0.01.

Statistical analyses were performed on SF sterility values, as well as on the data of zygotic and maternal and paternal effects, by using the Statgraphics Plus 3.1 software (Statistical Graphics Corporation). ANOVA was done to determine if the zygotic and maternal and paternal effect values—which are differences between two sterility levels (see text)—were statistically different from zero (at P ⬍ 0.01 or ⬍ 0.05).

RESULTS

Establishing transgenic lines: Various constructs expressing either antisense RNA (asRNAs) targeting the IF ORF1 and/or sense RNAs (sRNAs) corresponding to the same sequence were designed (Figure 1). Because germline expression of IF is restricted to females (Chaboissier et al. 1990; Lachaume et al. 1992; Seleme et al. 1999), asRNAs were driven by the promoter of the actin5C gene, which is strongly expressed during oogenesis (Fyrberg et al. 1983). sRNAs were produced from the weak internal IF promoter present in some constructs. To have the opportunity to use either I or R strains to deliver one, two, or three copies of each transgene to the SF females, the following procedure was followed. First, every independent transgenic insertion was established in the reactive strain wK as a homozygous line and assigned to a chromosome. Second, identical transgenes inserted on different chromosomes were combined to yield lines homozygous for two or three different insertions (materials and methods). Among these transgenic lines no variegation of the colored-eye phenotype associated with the mini-white selection marker was observed, suggesting that no transgenes were subject to a repressive effect such as those mediated by the Polycomb group of proteins or the modifiers of position effect variegation and that increasing copy number did not result in heterochromatinization of the transgenic

marker. Third, to have the set of lines in I and R states, the R transgenic lines were contaminated by functional IFs as previously described (Lachaume and Pinon 1993). Finally, Northern blot analysis performed on ovarian extracts from all transgenic lines confirmed that the transgenes strongly expressed transgenic asRNAs with the expected length. By contrast, sRNAs were faintly or not detected (not shown). The phenotypic effects observed with each construct are depicted in Figure 1, which gives a brief overview of the results discussed below. SF sterility results from IF mobilization: The effect of RZI, which produces asRNA from ⫹1 to ⫹1104, on IF expression and the sterile female phenotype was evaluated. The mere presence of this transgene, however, might modify the level of reactivity and its maternal transmission in the transgenic lines ( Jensen et al. 1995). To avoid this bias, RZI was transmitted from homozygous males. R males carrying the RZI construct were crossed to R females carrying the IF-lacZ construct (Figure 1) to give R female progeny, the ovaries of which were assessed for transgenic asRNAs and ␤-galactosidase without interference from naturally occuring IFs. The SF females, daughters of I RZI males crossed to R wK females, were checked in parallel for their sterility without interference from the IF-lacZ construct (Figure 2). Increasing the copy number of RZI increased the amount of asRNAs (Figure 2A). This clearly rules out any cosuppression between the various copies of RZI. Moreover, there was a correlation between the expression of RZI in R ovaries, as defined by the level of asRNAs, and (1) the IF expression level in R ovaries, as monitored by the IF-lacZ reporter, and (2) the degree of sterility of isogenic transgenic SF females without the IF-lacZ target. The increasing amount of asRNAs correlated with a decrease in both the amount of ovarian transgenic

Regulation of I Factor Transposition

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Figure 1.—Structure of the I factor and transgenes. Nucleotide positions are given according to Fawcett et al. (1986). IF, general organization of the I factor (IF ) with its two nonoverlapping open reading frames (not drawn to scale). IF transcription is driven by a weak internal promoter and is initiated at nucleotide ⫹1 (arrow). Functional and potential initiating codons are indicated. 290, 1014, and 1104 indicate, respectively, the BstBI, BstBI, and SspI restriction sites used for transgene construction. Zn-f indicates the zinc-finger domain also found within the gag domain of retroviruses. IF-lacZ is a translational fusion between IF and the ␤-galactosidase gene from Escherichia coli previously described in Lachaume et al. (1992); the Zn-f domain has been deleted (⌬). This construct is present at two homozygous loci in the previously described 4I-lacZ strain (Tatout et al. 1994). RZI contains part of the ORF1 of IF (from ⫹1 to ⫹1104) transcribed in antisense orientation from the act5C promoter and in sense orientation from the IF promoter. RZAP is similar to RZI, but contains three mutations: ⌬, a deletion of the first five nucleotides essential for IF promoter activity (McLean et al. 1993), and X, missense mutations of both ATG187 and ATG586 to TAG and CGT codons. 8S expresses from the IF promoter a truncated sense RNA (⫹1 to ⫹1104) that can be potentially translated from ATG187 or ATG586. All four insertions have been obtained on the same chromosome, and no line with multiple insertions has been constructed. BB produces an antisense RNA corresponding to the central part of ORF1 (⫹290 to ⫹1014). n, number of independent reactive lines established for each transgene. At the right of the figure the phenotypic effects recorded and detailed in the course of this study are summarized.

␤-galactosidase and the SF lethality level (Figure 2B). This correlation demonstrates a link between the expression level of IF and the appearance of SF sterility, the main phenotypic trait of hybrid dysgenesis. Detection and assessment of zygotic and parental repressive effects induced by RZI: Is RZI also able to induce a maternal effect on SF females in addition to the zygotic asRNA effect described above? To address this point, the mating schemes described in Figure 3 were used. The aim was to obtain simultaneously transgenic (Tg⫹, or colored-eyed) as well as nontransgenic (Tg⫺, or white-eyed) SF females derived from the same heterozygous parents. Their respective levels of sterility could be compared with that of an SF control, which had no transgene in its ancestry. The number of maternal generations through which the transgene was transmitted varies according to each protocol: n (Figure 3A), 1 (Figure 3B), or 0 (Figure 3C). If the level of sterility is lower in the Tg⫺ females than in control females, this rescue should be ascribed to the presence of the transgene in the previous generation(s)—that is, to a maternal effect [either accumulated over several generations (Figure 3A) or in one generation (Figure 3B)] or to a putative paternal effect (Figure 3C). In contrast, a zygotic effect is assessed by comparing the sterility levels of Tg⫹ and Tg⫺ sibling SF females when there is no maternal effect, that is, when the transgene is transmitted through males to SF females (Figure 3C). The RZI transgene induces zygotic, maternal, and paternal effects: Ten generations after transgene establishment in an R or I context, the various effects of one, two,

and three copies of RZI were evaluated. The sterility levels of the different SF females were checked daily during the first week of egg laying. Average values obtained each day from the two 3RZI lines are shown in Figure 4. The SF control exhibited high sterility, indicating that the reactivity level was not affected by our experimental conditions. However, after maternal transmission of the transgenes over several generations (Figure 4A), transgenic (Tg⫹) SF females exhibited full reversion of the sterility phenotype (⬍10% dead embryos) as early as the first day of egg laying. Strikingly, total repression of hybrid dysgenesis was also recorded in the nontransgenic SF females (Tg⫺). This proved the existence of a maternal repressive effect, induced only by the presence of the transgenes within the maternal genome. To test whether the maternal effect developed fully within a single generation, or increased over several generations, the protocol of Figure 3B was applied: three copies of RZI were transmitted through one maternal generation. The results (Figure 4B) show that a maternal effect was still observed in the Tg⫺ SF females, but that it was weaker: the effect was significant only from the fourth day of egg laying and it rescued even fewer embryos at day 7 (compare with Figure 4A). Thus the maternal effect induced by RZI is a quantitative effector that accumulates over generations. Paternal transmission (Figure 3C) could not be tested for three copies of RZI, because all lines had a transgene insertion on the chromosome X that is not transmitted from father to daughters; instead, the zygotic effect of two copies of RZI was evaluated. A pure paternal effect,

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Figure 2.—Correlations among asRNA production, IF expression, and SF sterility. First, reactive females carrying two homozygous copies of IF-lacZ (Tatout et al. 1994) were crossed with reactive males carrying zero to three homozygous copies of RZI. Reactive female progeny were kept for 5 days, and then their ovaries were dissected. Ovarian asRNA accumulation was evaluated by slot-blot analysis on poly(A)⫹ RNA extracts and the amount of bacterial ␤-galactosidase was estimated using ELISA tests (see materials and methods). Second, isogenic (but not transgenic) reactive females were crossed with inducer males carrying zero to three homozygous copies of RZI. SF progeny were kept until 5 days old and checked for their sterility. (A) asRNA accumulation [in nanograms of asRNA/␮g of poly(A)⫹ RNA] as a function of RZI copy number. (B) Bacterial ␤-galactosidase amount (in nanograms of ␤-galactosidase/milligrams of total ovarian protein, open triangle) and SF sterility (percentage of dead embryos, solid squares) as a function of RZI copy number.

distinct from a zygotic effect, was identified as the difference of repression between the Tg⫺ and control SF females, as the SF females aged (Figure 4C). This paternal inheritance of repression, which has not been previously described, proves that some inherited effectors of regulation can pass through male meiosis. Zygotic and maternal effects increased with the number of RZI copies, but whether the paternal effect follows the same rule was not as clear (Table 1). At first glance, zygotic and maternal and paternal effects seemed to be

additive; however, individual and precise evaluation of each effect became difficult (1) when individual values fell below 10% and lost statistical significance (for examples, see Table 1) or (2) when one effect reached a value ⬎80% and masked any other effect (in Figure 4A the accumulated maternal effect is so high that the zygotic effect—the difference between the Tg⫹ and Tg⫺ curves—of three copies of RZI can no longer be observed). In addition, all these effects were dynamic and varied as the SF females aged (Figure 4). To compare the various experimental conditions described here, the percentages of rescued embryos were combined within strains (from day 1 to 7) and over strains according to the transgene copy number (see materials and methods) to give the “average effect of the average line” (see Table 1, RZI). The paternal and maternal effects of RZI suggest that epigenetic mechanisms are involved in the regulation of hybrid dysgenesis by this transgene. However, none of the previous hypotheses (see Introduction) could be excluded: (1) a defective and repressive ORF1 protein that was accumulated from our construct; (2) a putative activator of IF expression that was depleted by our construct; or (3) an epigenetic phenomenon induced by the introduction of IF sequences into the Drosophila genome, whatever their expression abilities might be. To discriminate among these hypotheses, we analyzed the effects of three new constructs: RZAP, 8S, and BB (Figure 1). Zygotic effects are not induced by a defective ORF1 protein but by specific asRNAs: Construct RZAP was derived from RZI, but had no functional IF promoter and was further mutated in the initiation codons at 187 and 586 (legend to Figure 1). Construct BB expressed asRNA, like RZAP, but contained only the central part of ORF1 (from 290 to 1104). Construct 8S was derived from RZI but had no actin5C promoter. Table 1 shows that RZAP and RZI induced similar zygotic effects, but 8S and BB did not. Results with BB were unexpected; this construct expressed asRNA but none of the 16 transgenic lines ever tested positive for a zygotic effect. In fact, RZAP and RZI expressed the same asRNAs, while BB expressed shorter asRNAs that hybridized with 724 bases of their target, but neither with the 5⬘-untranslated region (UTR), nor the ATG187 region, nor the 1014–1104 segment of IF (Figure 1). This suggests that any of these parameters, or some combination of them, is required to develop a zygotic effect. 8S contains the same IF sequences as RZAP and RZI, but it did not express asRNA; its lack of a zygotic effect confirms, a contrario, that the zygotic effect observed with RZI is due to asRNAs longer than those produced by BB. It also rules out the hypothesis that a truncated protein encoded by ORF1 in RZI accounts for the zygotic and paternal effects. Genetic features of the maternal effect: A maternal


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Figure 3.—Mating schemes used to discriminate the zygotic antisense effect from parental effects. Transgenes were followed by the colored-eye phenotype they confer. Tg, transgene (one, two, or three copies); control, isogenic but nontransgenic strain; ⫹, wild-type nontransgenic chromosome (for clarity a single autosomal chromosome is shown); I, inducer strain; R, reactive strain; SF, dysgenic sterile female. In all crosses SF females were isogenic except for the transgenic insertions. The long-term (A) and short-term (B) maternal effects were investigated by using Tg/⫹ females as mothers of SF females; transgenes were transmitted through either n maternal generations (A) or one maternal generation (B) before the dysgenic cross. The maternal effect was evaluated by subtracting the percentage sterility of Tg⫺ SF females from the percentage sterility of control SF females in both crosses. (C) Zygotic effect and paternal effect were investigated using Tg/⫹ males as fathers of SF females. The zygotic asRNA effect was evaluated by subtracting the percentage sterility of Tg⫹ SF females from the percent-sterility of their Tg⫺ SF sisters. The paternal effect was evaluated by subtracting the percentage sterility of Tg⫺ SF females from the percentage sterility of control SF females.

effect was detected for all constructs—except for BB— and it increased with the copy number of the transgene and was additive with the zygotic effect (Table 1). One construct, 8S, exhibited only a maternal effect, an effect also developed by the IZ transgenes of Jensen et al. (1995) that contain a complete ORF1 (from nucleotide 187 to 1464, compared to 187 to 1104 for 8S). Thus a full-length ORF1 was not required to induce the maternal repressive effect. However, the fact that BB had no maternal effect indicated either that the 5⬘-UTR (and/ or the 1014–1104 part of ORF1) of the IF was responsible

for the maternal effect or that the BB construct, despite its 724 bp from IF, was not long enough to mediate such a maternal effect. Initial quantitative analysis showed that the maternal effect increased with strain aging. The stability of all effects was therefore tested. In fact, the efficiency of the maternal effect increased after transgene establishment, over at least 100 generations for RZI, while the zygotic and paternal effects remained constant (Figure 5). The kinetics suggest that this increase has no other boundary than the rescue of all embryos laid by SF females. This

Figure 4.—Inhibition of SF sterility by RZI transgenes. Ten generations after establishment of the RZI lines, the phenotypic effect of three copies of RZI was evaluated according to mating schemes A and B in Figure 3. Transmission of only two copies of RZI was analyzed through zero maternal generation (mating type C, Figure 3), as paternal transmission loses the insertion on the X chromosome. SF sterility was recorded daily and in triplicate over the first week of egg laying. Results are expressed in percentage sterility (see materials and methods). Control SF females, dashed lines; solid symbols, transgenic (Tg⫹) SF females; open symbols, nontransgenic (Tg⫺) SF females. Shaded areas, maternal effects (A and B) and paternal effect (C).

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Figure 5.—Evolution of zygotic, maternal, and paternal effects as a function of strain aging. The effects of a single copy of transgene were evaluated 10, 20, 50, and 100 generations after transgene establishment using protocols in Figure 3, A and C. The evaluation was repeated three times in parallel for each line. Average values from all the lines at our disposal are expressed in percentage of embryos rescued by zygotic (hatched box), maternal (open box), or paternal (solid box) effect.

cumulative property is also shared, to a lesser extent, by reactivity, but distinguishes the maternal effect from the paternal effect. The stability of the maternal effect was evaluated one, two, or three generations after transgene removal (Figure 6). Two generations after removal, the repressive effect was still evident (ⱖ20% of all embryos rescued). At the third generation, however, repression of hybrid dysgenesis was significant (rescue ⬎ 10%; P ⬍ 0.05) only for the maternal effect induced by two or three copies of the transgene: 15 ⫾ 5, 18 ⫾ 7, and 19 ⫾ 9% of maternally rescued embryos over the week when derived from the 2RZI, 3RZI, and 2RZAP lines, respectively (Figure 6, B and C). However, when only the 6th and 7th days of egg laying were considered, the maternal

Figure 6.—Perdurance of the maternal effect of each transgene. The maternal effect was evaluated for all transgenic lines in Tg⫺ SF females, as shown in A. Sterility of Tg⫺ SF females was recorded during the first week of egg laying, when the reactive mother was heterozygous for the transgene (G0), or one, two, or three generations after transgene removal (G1, G2, and G3, respectively). At each generation, the percentage sterility of these Tg⫺ SF females was compared to the percentage sterility of an SF control, as in Figure 3. (B) Perdurance of maternal effect induced by one, two, or three copies of RZI. (C) Perdurance of maternal effect induced by one or two copies of RZAP, or one copy of 8S.

effect induced by a single copy of the transgene was higher than 10% at P ⬍ 0.05 (not shown). Altogether, these results demonstrated a surprisingly long-lasting memory of the maternal effect after transgene removal. Paternal effect, maternal effect, and reactivity: The paternal effect was induced by RZI and RZAP, but was markedly weaker than the maternal effect (Table 1). In fact, it was never significant before the 5th day of egg laying (Figures 4C and 7), which may explain why it has never been described before; however, the paternal


Figure 7.—Effect of SF female aging on the paternal effect, maternal effect, and reactivity. Dashed line, evolution of the reactivity level of SF females as a function of their aging (average value from 16 independent experiments). Continuous lines: maternal (m, solid symbols) and paternal (p, open symbols) effects of a single copy of RZI, RZAP, 8S, and BB, as a function of aging in young SF females. Data are from lines arbitrarily chosen 20 generations after transgene establishment.

effect induced by two copies of the transgenes could rescue as many as 40% of all embryos laid by SF females on the 7th day of laying. The very existence of such an effect supports previous hypotheses on the involvement of epigenetic mechanisms in the regulation of IF. The paternal effect is, however, different from the maternal effect: (1) it is not affected by strain aging (above and Figure 5); (2) it appears only in lines already exhibiting a zygotic effect; and (3) it is not detected in the 8S lines even though they exhibit a maternal effect (Table 1). Thus the paternal effect is not just the mirror image of the maternal effect in a different meiosis. However, as a noncytoplasmic mechanism, it might be a component of the maternal effect. The paternal effect increased with female aging, like the maternal effect, while reactivity decreased (Figure 7). The apparently increasing efficacy of RZI, RZAP, and 8S during female aging might, then, be a consequence of this decrease of reactivity, while the actual transgene efficacy remained constant. DISCUSSION

IF mobilization is responsible for the hybrid dysgenesis trait: AsRNAs were used previously to demonstrate the involvement of the P element in P-M hybrid dysgenesis (Simmons et al. 1996). Here, this approach was used to demonstrate the involvement of another transposable element, the IF, in I-R hybrid dysgenesis. First, when the RZI and RZAP transgenes are inherited from I males, the zygotic component of the repressive effect reflects this “antisense” targeting of the IF transcripts within the germline of SF females, since 8S does not exert any zygotic repressive activity. Second, the comparison be-

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tween RZAP and 8S clearly demonstrates that asRNAs are able to downregulate IF expression during oogenesis. Third, a comparison of RZAP and BB suggests that asRNAs must hybridize with the 5⬘-UTR and/or AUG187 and/or the 1014–1104 segment of the IF transcript to be fully effective. Fourth, the higher antisense efficiency of RZI compared to RZAP may be a consequence of dsRNA formation in the case of RZI, since dsRNAs have been shown to have efficient repressive properties in Drosophila as well as in Caenorhabditis elegans through a process called “RNA interference” (Fire et al. 1998; Kennerdell and Carthew 1998; Hammond et al. 2000). How is IF responsible for SF lethality? Is it through expression of IF proteins? Is it a consequence of insertional mutagenesis on the host genome? After a dysgenic cross, the IF invades the R genome. During this process IF is expressed in the female germline for 1 to 10 generations (Lachaume and Pinon 1993), and the number of IFs increases until it reaches 10 to 15 copies per haploid genome. Sterility, however, can be recorded for many more generations (Pe´lisson and Bre´gliano 1987). Furthermore, overexpression of IF-encoded proteins in transgenic flies is not lethal ( Jensen et al. 1995; Busseau et al. 1998), even though ORF2 encodes a putative DNA endonuclease known to be active in the related LINE L1Hs (Feng et al. 1996). These data suggest that IF expression, insertional mutagenesis, and DNA breaks induced during retrotransposition of the IF in ovaries cannot induce a high rate of SF sterility. Alternatively, IF insertions in chromosomal breaks arising at the beginning of meiotic recombination may induce a VAMOS response with lethal potential (Laurençon et al. 1997). If so, IF mobilization may then be considered one of the many signals that can activate the VAMOS recombination-repair system in the female germline of D. melanogaster. This may be similar to the induction of the SOS system during Tn10 transposition (Roberts and Kleckner 1988). Regulation of the IF: involvement of epigenetic mechanisms: IF sequences are strong mediators of their own repression through sense and antisense transcription (Jensen et al. 1999a,b; this work). However, asRNA-producing constructs exhibit more than a zygotic effect: repression of I-R hybrid dysgenesis by the RZI and RZAP transgenes reveals additional maternal and paternal controls on IF mobilization. The maternal effect induced by IF sequences is not only efficient but also accumulates for ⵑ100 generations and still exists two generations after transgene removal. The paternal effect is transmitted by sperm devoid of the transgene. These results argue for epigenetic control of IF. On the one hand, if maternal and paternal effects are related mechanistically, then at least part of the maternal effect is not obtained through accumulation of a regulatory molecule in the cytoplasm of the oocyte. On the other hand, reactivity is also able to vary slowly

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over generations through a cumulative maternal effect (Bucheton and Picard 1978) and may share a mechanistic basis with the parental effects described here. Maternally inherited effects have already been reported in Drosophila for epigenetic controls such as those mediated by the Polycomb group (Cavalli and Paro 1998). Paternal imprinting has also been described for the enhancer of the position effect variegation gene, E(var)393D (Dorn et al. 1993). Such a possibility is further strengthened by the fact that the IF-lacZ transgene inserted at euchromatic loci exhibited a variegated pattern of expression during oogenesis (Tatout et al. 1994). All these results, as well as the possible involvement of (1) IF promoter in transcriptional silencing (Chaboissier et al. 1998) and (2) IF transcripts in post-transcriptional silencing (Jensen et al. 1999a,b), point toward epigenetic mechanisms as key effectors in the control of IF transposition and autoregulation. This raises several prospective questions. What are the molecular mechanisms that are able to trigger and establish such a repression? How is this repression maintained and transmitted? Setting up the maternal effect through nucleic acid pairing? Length, transcriptional ability, and features of transgenes are important parameters in the setting of the maternal effect. Transgenes with various lengths of IF (from 186 bp to 1.5 kbp) have been tested ( Jensen et al. 1995, 1999a,b; Chaboissier et al. 1998; this work) with a wide range of efficacies: the shorter they are, the less effective they are. This might indicate that hybridization of nucleic acids is involved in the process. Indeed, Jensen et al. (1999b) propose that the maternal effect is induced by the presence of IF sense transcripts, which in turn induces a phenomenon analogous to cosuppression in plants (Matzke and Matzke 1995). If the direct involvement of euchromatic transgenic sequences in hybridization still has to be proven, there are several limitations to the use of RNA as sole effector of this repression. First, abundant IF sense transcripts already exist in Drosophila; they are encoded in all strains by defective elements located in pericentromeric heterochromatin (Vaury et al. 1990; Bucheton et al. 1992). Second, impairing sense transcription, as with the RZAP construct, should suppress the maternal effect, which it does not. A residual promoter activity might still exist in RZAP, but then it is difficult to understand why this construct has such pronounced maternal and paternal effects. This may be achieved through the production of aberrant RNAs from cryptic and adjacent promoters; such RNAs, like chimeric RNAs, have already been shown to mediate silencing (Mette et al. 1999; reviewed in Wassenegger and Pelissier 1998). Involvement of DNA sequences, through length and genomic copy number, could be consistent with a mechanism implicated in the control of dispersed repetitive elements like the IFs. Curiously, specific features of IF are required. Deletion of the 3⬘ end of ORF1 from nucleotide 1104 to 1464 does not abolish the maternal effect (this work: RZI, RZAP, 8S), whereas further dele-

tion (BB) of the 5⬘ end (from 1 to 290) and 3⬘ end (from 1014 to 1104) does. Transgenes limited to the untranslated leader of IF (from 1 to 186) have very low but still significant efficacy (Chaboissier et al. 1998), which suggests that the 5⬘ end of IF is one of many segments able to interact with functional IFs to induce silencing. Maintenance of the silencing: Transgenes do not need to interact directly with the functional IFs in SF females to repress I-R hybrid dysgenesis: (a) paternal and maternal effects are observed in SF females without the transgene, and (b) two generations without the transgene decrease but do not abolish the maternal effect. If these effects are associated with a local change in chromatin structure at the IFs insertion points, they should be “remembered” through mitoses and meioses after transgene(s) removal. One attractive hypothesis is that the maternal and paternal effects rely on the defective IF elements located at the pericentric heterochromatin to maintain the effects initiated by the transgenes. Strikingly, it has been shown that a heterochromatic P-element P1A, located near the telomere of the X chromosome, interacts with euchromatic P elements and induces trans-silencing (Ronseray et al. 1996; Roche and Rio 1998). In fact, endogenous DNA sequences might be key mediators in homology-dependent gene silencing (Pal-Bhadra et al. 1999). Thus, (1) if transgenes such as RZI or RZAP are able to change the chromatin structure of the pericentric defective I elements, and (2) if these changes modify the level of expression of functional IFs even after transgene removal, it would be interesting to assess the effect of mutations affecting the trans-silencing of P elements. Involvement of the population of defective heterochromatic IFs in the regulation of euchromatic IFs might also explain the particular inheritance of reactivity. Whatever the precise mechanism by which IF repression is achieved, the growing evidence that transposable elements are regulated through epigenetic phenomena opens a great field of investigation for an unsolved problem: how the host genome deals with advantages brought by transposable elements despite their tendency to invade and mutate the genome. Epigenetic effects might then be viewed as a real buffer against the massive expansion of transposable elements in eukaryotic genomes. We thank R. M. Molina for her helpful assistance in performing statistical analysis. This work was supported by University Claude Bernard—Lyon1, and by grants 6577 and 9856 from “Association pour la Recherche contre le Cancer” (ARC). E.G. received a fellowship from the Ministe`re de l’Enseignement Supe´rieur de la Recherche et des Technologies (MESRT). C.T. received a fellowship from the European Community through the “Molecular Tools for Biodiversity” fund.

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