Gene Silencing Triggered by Non-LTR Retrotransposons in the Female Germline of Drosophila melanogaster

Corresponding author: Isabelle Busseau, CNRS, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France., busseau{at}igh.cnrs.fr (E-mail)

Communicating editor: M. J. SIMMONS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.

TRANSPOSABLE elements, which are repetitive sequences capable of moving in the genome, are widespread in eukaryotes. Their transposition results in mutations and genome rearrangements that may be deleterious to the host, but also may have beneficial effects over evolutionary time when occurring in the germline. It is generally accepted that repressive mechanisms that prevent unconstrained transposition, while still allowing it to occur at very low levels, have evolved (YODER et al. 1997 ). Early hypotheses suggested the involvement of repressor proteins encoded by the elements themselves. These repressor proteins would accumulate and block further transposition when a certain number of copies of the element is reached. Such hypotheses are now clearly inadequate in most, if not all, cases. Recent data accumulated in various organisms, including Drosophila, strongly suggest that the activity of transposable elements is sensitive to host processes controlling repeated sequences such as RNA interference (KETTING et al. 1999 ; TABARA et al. 1999 ; ARAVIN et al. 2001 ; STAPLETON et al. 2001 ) and modifications of chromatin structure (LINDROTH et al. 2001 ; BIRD 2002 ; JACKSON et al. 2002 ; TOMPA et al. 2002 ). Whatever the molecular mechanism, it is supposed to be triggered by the repetitive nature, rather than by some specific intrinsic property, of the transposable elements.

All major classes of transposable elements, each represented by several families, exist in the genome of Drosophila melanogaster (KAMINKER et al. 2002 ). Many of these families contain potentially active members, capable of transposition. However, transposition is usually a very rare event, suggesting that active members are constitutively kept silent. Noticeable exceptions are the DNA transposons P and Hobo and the non-LTR retrotransposon I, which can be induced to transpose at high frequencies in the progeny of certain crosses, a phenomenon known as hybrid dysgenesis (for reviews see BLACKMAN et al. 1987 ; BUCHETON et al. 2002 ; RIO 2002 ). These particular cases provide ideal situations to investigate the mechanisms underlying the repression and activation of transposable elements, and recent investigations have revealed the possible involvement of homology-dependent gene-silencing mechanisms in some aspects of the regulation of the P and the I elements (CHABOISSIER et al. 1998 ; JENSEN et al. 1999A , JENSEN et al. 1999B ; GAUTHIER et al. 2000 ; MALINSKY et al. 2000 ; MARIN et al. 2000 ; RONSSERAY et al. 2001 ).

The work presented below focuses on two D. melanogaster non-LTR retrotransposons, R1Dm and I. Both share a similar structure with two open reading frames (ORFs), the second of which contains conserved endonuclease and reverse transcriptase domains. R1Dm is a highly sequence-specific element that inserts at a precise position, and always in the same orientation, in the 28S gene of the tandemly repeated rDNA units located on the X chromosome (JAKUBCZAK et al. 1990 ). Depending on the strain, between 23 to 60% of the 150 or so rDNA repeats are found to contain an R1Dm insertion (JAKUBCZAK et al. 1992 ). It is assumed that R1Dm elements are devoid of a promoter and are cotranscribed along with the ribosomal units into which they are inserted, producing chimeric 28S/R1Dm RNAs that serve as templates for retrotransposition. However, ribosomal genes that are interrupted by R1Dm insertions produce very little RNA: they are very poorly transcribed, and when they are transcribed the RNA becomes prematurely processed or degraded during transcription (LONG and DAWID 1979 ; LONG et al. 1981 ; JAMRICH and MILLER 1984 ). Consistent with this, retrotransposition of R1Dm occurs constitutively at low level (PEREZ-GONZALEZ and EICKBUSH 2002 ).

Unlike R1Dm, I elements insert randomly in the genome. The I element family is peculiar because active members, called I factors, invaded the genome of the species less than one century ago (BUCHETON et al. 1992 ). As a result of this recent invasion, all D. melanogaster strains fall into one of two categories: inducer, whose members contain a limited number of euchromatic copies of I, and reactive, whose members derive from flies that escaped the invasion (BUCHETON et al. 1992 ). I factors are repressed and remain stable in inducer strains, where transposition occurs very rarely. They transpose very efficiently in the germline of hybrid females, called SF females, resulting from crosses involving females from a reactive strain and males from an inducer strain. Similarly, when a single I factor is introduced by P-mediated transgenesis into the genome of a reactive line, it transposes actively. In all cases, after a few generations the number of copies reaches a certain point above which transposition stops, and the strain becomes inducer (PICARD 1978 ; PRITCHARD et al. 1988 ; ABAD et al. 1989 ; VAURY et al. 1993 ). The number of euchromatic copies of I in an inducer strain is difficult to estimate due to the frequent generation of defective copies, usually truncated at the 5' end, in the course of retrotransposition. The number of active I factors is thought to be around five (PELISSON and PICARD 1979 ). Analysis of the sequenced euchromatin of an inducer strain of D. melanogaster has identified 28 I elements among which 8 are full length (KAMINKER et al. 2002 ). A key molecule in the mechanism of retrotransposition of the I factor is the full-length RNA that is synthesized from an internal female germline-specific polII promoter, contained in the 5' untranslated region (UTR; CHABOISSIER et al. 1990 ; MCLEAN et al. 1993 ; CHAMBEYRON et al. 2002A , CHAMBEYRON et al. 2002B ). This full-length RNA is believed to act both as the messenger for translation of the two open reading frames and as the retrotransposition intermediate (CHABOISSIER et al. 1990 ; BOUHIDEL et al. 1994 ). It accumulates in the female germline only in conditions allowing high retrotransposition. Its abundance is correlated with the transposition frequency. It is not detected in the germline of inducer females. Thus, the regulation of I factor retrotransposition in the female germline is expected to act on the accumulation of the full-length RNA molecule at either the transcriptional or the post-transcriptional level.

The starting point of our work was the observation that chimeric I elements that contain the endonuclease domain of R1Dm in place of their own do not produce detectable transcripts in the female germline. This observation prompted us to analyze, in the female germline of D. melanogaster, the silencing effect of various sequences from non-LTR retrotransposons. To this end, we designed female germline-specific chimeric transcription units, inserted into these units various sequences to be tested as silencers, and analyzed their expression by in situ hybridization experiments on ovaries. Our data indicate that transcription units containing parts of the R1Dm or I non-LTR retrotransposons of D. melanogaster can be silenced in the female germline and that this silencing occurs only in the presence of multiple endogenous genomic copies of the corresponding element. Thus, while previous reports had shown that non-LTR retrotransposons like I elements are sensitive to homology-dependent gene-silencing mechanisms (CHABOISSIER et al. 1998 ; JENSEN et al. 1999A , JENSEN et al. 1999B ; GAUTHIER et al. 2000 ; MALINSKY et al. 2000 ), here we provide evidence that they can also trigger these mechanisms, thereby emphasizing the involvement of such mechanisms in their self-regulation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmid constructs:
All PCR amplifications used in plasmid constructions were performed using the Pfu DNA polymerase (Stratagene, La Jolla, CA) following the manufacturer's instructions and all constructs were checked by sequencing.

IER1Dm, IER1'Dm, and INuc: Starting at the beginning of ORF2, the rightmost part of an R1Dm element inserted in rDNA 28S was amplified by PCR from D. melanogaster genomic DNA using primers IR1-5' (5'-AAAGCTTTTAATTTTCTCACAAATGTTTAGCTTCATCCAAGCGAACTGT-3') and Eco-28S (5'-CTTGAATTCGATCATACCTGAGTAATTGG-3'). This PCR product was used as template in a second PCR amplification using primers IR1-5' and IR1-3' (5'-AACTCGAGAATACGTAAAGCGCAGAGGTCGGCAGTCCACCAACGTGCTCT-3'). The HindIII-XhoI fragment from this PCR product, containing the endonuclease domain of R1Dm (positions 1733–2663 in GenBank accession no. X51968), was cloned into the pET-21b vector (Novagen) to produce pET21b-ENDm. To construct IER1Dm, a SalI-HindIII fragment (positions 1–1615 in GenBank accession no. M14954) from pI954 (PRITCHARD et al. 1988 ) was introduced into SalI/HindII-cut pET21b-ENDm; then an EcoRI-SnaBI fragment from the resulting construct and a SnaBI-BamHI fragment (positions 2518–5671 in GenBank accession no. M14954) from phsORF2HN (BUSSEAU et al. 1998 ) were ligated together and inserted between the EcoRI and BamHI sites of pBluescript KS- (Stratagene) and of pCaSpeR-4 (THUMMEL and PIRROTTA 1992 ). To construct IER1'Dm, a PCR fragment containing the endonuclease domain of R1Dm (positions 1733–2420 in GenBank accession no. X51968) amplified from pET21b-ENDm using primers IR15' and NR1Xho (5'-AACTCGAGGCGCGCGGTAGTTGGTTCGGCCAC-3') and a PCR fragment (positions 2285–2525 in GenBank accession no. M14954) amplified from pI954 using primers Iup-BssH2 (5'-ACCGCGCGCAATCCACAAAAATTCTACAGACCC-3') and Ido-SnaBI-XhoI (5'-AACTCGAGTTTACGTAATTGGTCGAGGTG-3') were cut with BssHII and ligated together, reamplified using primers IR15' and Ido-SnaBI-XhoI, cut with HindIII and XhoI, and inserted into HindII/XhoI-cut pET21b. The resulting construct was then cut with SalI and HindIII and ligated to a SalI-HindIII fragment (positions 1–1615 in GenBank accession no. M14954) from pI954; then an EcoRI-SnaBI fragment from the resulting construct and a SnaBI-BamHI fragment (positions 2518–5671 in GenBank accession no. M14954) from phsORF2HN were ligated together and inserted between the EcoRI and BamHI sites of pBluescript KS- and of pCaSpeR-4. INuc was derived from IER1'Dm inserted in pBluescript KS- by deleting the HpaI-SnaBI fragment encompassing the endonuclease domain and reintroduced in pCaSpeR-4 in an EcoRI-BamHI fragment.

IH and derivatives: IH is a SalI-HindIII fragment (positions 1–1615 in GenBank accession no. M14954) from pI954 and a HindIII-SmaI fragment (positions 2638–5473 in GenBank accession no. M14954) from pITK (CHAMBEYRON et al. 2002B ) ligated together and inserted between the SalI and SmaI sites of pBluescript KS-. IH was reintroduced into pCaSpeR-4 as an XhoI-HindIII fragment. The resulting construct was cut with NheI and ligated with relevant XbaI-cut PCR products to generate IH-O1Dm, IH-EDm, IH-O2Dm, IH-EBm, and IH-LacZ derivatives.

yema-LacZ and derivatives: yema-LacZ was derived from construct D (CAPRI et al. 1997 ) by replacing the BamHI fragment containing the entire ß-galactosidase gene with a 900-bp PCR fragment, LacZ₉₀₀, amplified from the ß-galactosidase gene (positions 23–926 in GenBank accession no. V00296) using primers LacB-up (5'-AAGGGGGATCCCGTCGTTTTAC-3') and Lac-XB-do (5'-CTTGGATCCTCTAGAGATTCGGGATTTCGGCGC-3'). The construct was cut with XbaI and ligated with relevant XbaI-cut PCR products to generate yema-LacZ-EDm and yema-LacZ-O1I derivatives.

PCR fragments: O1Dm, EDm, and O2Dm (positions 389–1158, 1770–2658, and 3067–3688, respectively, in GenBank accession no. X51968) were amplified from the R1Dm element cloned in plasmid 5,6kb-H3 (JAKUBCZAK et al. 1990 ) using, respectively, primers XbaI-ORF1R1Dm-up (5'-GCTCTAGAGCGACAGCAGTGTGAGTGCCT-3') and XbaI-ORF1R1Dm-do (5'-GCTCTAGAGTACGAATGATCGCACCACCA-3'), ENR1-Xba-up (5'-GCTCTAGAGCTGCGACCATCGAGCTCGG-3') and ENR1-Xba-do (5'-TATCTAGAGGTCGGCAGTCCACCAACG-3'), and XbaI-ORF2R1-Dm-up (5'-GCTCTAGAGCAGGCGCTCTCCCGGG-3') and XbaI-ORF2R1Dm-do (5'-GCTCTAGACGCTGAAGCAGTACATCCATCAGTATG-3'). EBm (positions 1881–2544 in GenBank accession no. M19755) was amplified in two steps: first, the rightmost part, starting at the beginning of ORF2, of an R1Bm element inserted in rDNA 28S of Bombyx mori, was amplified using primers XbaI-ENR1Bm-up (5'-GCTCTAGAATGGATATTAGGCCCCGACTTCG-3') and Eco-28S-Bm-do (5'-GCGAATTCCGCCACGTCACCACTCTG-3'). This PCR product was then used as template in a second PCR amplification using primers XbaI-ENR1Bm-up and XbaI-ENR1Bm-do (5'-GCTCTAGATGTACCGCCCCCCACCCCAAA-3'). LacZ₈₀₀ contained in IH-LacZ was amplified from the ß-galactosidase gene (positions 120–926 in GenBank accession no. V00296) using primers XbaI-LacZ-up (5'-GCTCTAGAGGCCCGCACCGATCGCCC-3') and Lac-XB-do (5'-CTTGGATCCTCTAGAGATTCGGGATTTCGGCGC-3'). O1I was amplified from pI954 (positions 291–1113 in GenBank accession no. M14954) using primers ORF1IXba-up (5'-GCTCTAGACCCACCAAACATTTACAAAATC-3') and ORF1IXba8-do (5'-TATCTAGAAATATATGTGTTGGGCCG-3').

D. melanogaster stocks and transgenic lines:
All flies were kept at 22–23°. The standard strong reactive strain used in all experiments was JA (y w). The JAI2 (y w) inducer line is isogenic to JA except for the presence of transposed copies of the I factor. It was derived from dysgenic females obtained from crosses of females from the JA strain with males containing a single active I factor [I954Y in transgenic line 57.3 (CHABOISSIER et al. 1998 , CHABOISSIER et al. 2000 )]. These dysgenic females were highly sterile due to a high level of I retrotransposition. They were crossed with JA males and allowed to lay eggs for several weeks until the hatching of a few of their eggs. Resulting adults were mated together, and their white-eyed progeny (devoid of the original I954Y transgene) were selected to produce the subsequent generation from which JAI2 was established.

P-element-mediated transformations (SPRADLING and RUBIN 1982 ) were performed using pUChsP2-3 (FlyBase no. FBmc0002087) as the source of transposase. The recipient strain was JA, and homozygous transgenic lines were established by selecting dark-orange-eyed flies. Isogenic inducer transgenic lines were established by crossing JAI2 females with males of transgenic lines and by selecting dark-orange-eyed flies in subsequent generations. Inducer transgenic lines were maintained for at least 11 generations before being used in the experiments.

In situ hybridization on whole-mount ovaries:
RNA detection by in situ hybridization on whole-mount ovaries was performed following the protocol of TAUTZ and PFEIFLE 1989 adapted by CAPRI et al. 1997 . Probes were PCR products amplified using the Taq Polymerase (Promega, Madison, WI), purified using the QIAquick PCR purification kit (QIAGEN, Chatsworth, CA), and labeled using the Dig-Nick translation mix (Boehringer Mannheim, Indianapolis). Probe I186 is a PCR fragment corresponding to the I factor 5' UTR (positions 100–285 in GenBank accession no. M14954) amplified from pI954 using primers NotI-Iup-809 (5'-TAAAGCGGCCGCTATGCAAATCAGTACCACTTC-3') and Ido-1003-Hind3 (5'-CACGAAGCTTGATTGTTGGTTAAGGGCTT-3'). Probe yem is a PCR fragment corresponding to positions 3272–4089 in GenBank accession no. X63503 and amplified from pygA1 (AIT-AHMED et al. 1987 ) using primers OA65 (5'-AACTATGAGACGGAACTG-3') and OA70 (5'-GCTAGGGGATGCCAGATC-3'). Probes LacZ₈₀₀ and O1I are described above.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Chimeric non-LTR retrotransposons reveal gene silencing in the female germline:
Our aim initially was to analyze the behavior of chimeric non-LTR retrotransposons. We constructed chimeric elements based on the I factor, in which a region at the 5' end of ORF2 was replaced by the homologous region from the R1 retrotransposon of D. melanogaster (R1Dm). Two chimeras were designed (Fig 1). In IER1Dm, the replacement involves the region corresponding to the putative endonuclease domain of ORF2 in I and R1 (FENG et al. 1996 , FENG et al. 1998 ; MARTIN et al. 1996 ) and some additional sequences toward the 3' end of this domain. In IER1'Dm, the substitution involves only the putative endonuclease domain. The two chimeras were introduced into the genome of the JA reactive strain by P-element-mediated transformation. Analyses of several independent transgenic lines failed to detect any retrotransposition event of the chimeras (data not shown).

View larger version (0K): In this window In a new window Download PPT slide   Figure 1. Structures of the I factor, R1Dm, and chimeric elements. Narrow boxes represent untranslated regions and wide boxes represent open reading frames: white for I factor sequences, shaded for R1Dm sequences. , the PCR fragment I186 used as probe in in situ hybridization experiments. The positions of relevant HpaI (Hp) and SnaBI (S) restriction sites are indicated.

The two chimeras possess all known sequences required for efficient transcription of the I factor (MCLEAN et al. 1993 ) and correct localization of the transcripts in the ovaries (M. C. SELEME, D. TENINGES and A. BUCHETON, unpublished data) in a reactive background. They were therefore expected to produce high levels of transcripts in the oocytes, easily detectable by in situ hybridization on whole-mount ovaries using an I factor probe (BUCHETON et al. 2002 ; CHAMBEYRON et al. 2002B ). Surprisingly, no transcripts of the IER1Dm and IER1'Dm chimeric elements could be detected in the ovaries of transgenic females (Table 1 and Fig 2).

View larger version (0K): In this window In a new window Download PPT slide   Figure 2. Silencing of chimeric elements. Typical in situ hybridization experiments on ovaries of the JA strain (a) and of transgenic females containing IER1Dm (b), IER1'Dm (c), and INuc (d). The probe is I186 (Fig 1). Arrows show some examples of RNA detection in the oocyte.

To ascertain that the silencing of the chimeras is not due to the lack of important sequences of the I factor, we deleted a HpaI-SnaB1 fragment encompassing the R1-originating sequences from the IER1'Dm chimera (Fig 1). The resulting construct, INuc, was introduced into the genome of the JA reactive strain by P-element-mediated transformation. In situ hybridization experiments on ovaries of transgenic females show that this element produces transcripts localized as those of active I factors (Table 1 and Fig 2). Therefore, it is the presence per se of sequences of the 5' end of ORF2 of R1Dm in the IER1Dm and IER1'Dm chimeras that results in the absence of transcripts.

Silencing of a chimeric I element by R1Dm sequences:
We designed the IH element, which derives from the I factor by a deletion of the 1-kb fragment between the two HindIII sites (Fig 3). It is therefore very similar to the INuc element. It contains a unique NheI site allowing easy introduction of DNA fragments to be tested for silencing effects.

View larger version (0K): In this window In a new window Download PPT slide   Figure 3. Structures of chimeric transcription units. Legend is the same as for Fig 1. The different PCR fragments used in this study are indicated by solid boxes below the elements and constructs. (a) Structure of the I factor and of the IH element. (b) Structure of the R1Dm and R1Bm elements. (c) Structure of the yemanuclein- gene and of the yema-LacZ construct. The diagonally hatched boxes represent sequences of the yemanuclein- gene; introns are indicated as breaks in the boxes. The dotted box represents sequences from the bacterial ß-galactosidase gene, and the vertically striped box represents the 3' untranslated region of SV40. The positions of relevant HindIII (H), NheI (N), and XbaI (X) restriction sites are indicated.

Three different fragments, O1Dm, Edm, and O2Dm, from various regions of R1Dm (see Fig 3) were generated by PCR on genomic DNA of the JA strain using specific pairs of primers designed to create, at both ends, an XbaI site compatible with NheI. Similarly, an 800-bp fragment, LacZ₈₀₀, from the bacterial ß-galactosidase gene was also generated. These fragments were introduced into the NheI site of IH. The resulting constructs, IH-O1Dm, IH-EDm, IH-O2Dm, and IH-LacZ (Fig 3), were introduced into the genome of the JA reactive strain by P-element-mediated transformation. Several transgenic lines were established for each construct, and in situ hybridization experiments were performed on ovaries of transgenic females. The results are summarized in Table 1, and typical data are shown in Fig 4. The IH and IH-LacZ elements produce detectable transcripts in the oocyte (Fig 4, a and b) as do active I factors in SF females (BUCHETON et al. 2002 ; CHAMBEYRON et al. 2002B ). By contrast, none of the transgenes containing IH-EDm produce detectable transcripts (Fig 4G), as expected from the results described above. The majority of the transgenes containing other parts of R1Dm, IH-O1Dm and IH-O2Dm, do not produce detectable transcripts as well (Fig 4C and Fig E). Some of them produce transcripts but at a visibly lower level than IH (Fig 4D and Fig F).

View larger version (0K): In this window In a new window Download PPT slide   Figure 4. Silencing of IH by R1 sequences. Typical in situ hybridization experiments on ovaries of transgenic females containing IH and its derivatives. The probe is I186 (Fig 1). (a) IH; (b) IH-LacZ; (c and d) IH-O1Dm; (e and f) IH-O2Dm; (g) IH-EDm; (h) IH-EBm. Arrows show some examples of RNA detection in the oocyte.

Thus, independent regions of R1Dm silence IH when they are present within the element, whereas sequences from the bacterial ß-galactosidase gene do not. It is very unlikely that this silencing effect is due to three specific transcriptional silencing elements present in the DNA of R1Dm. Rather, these results suggest that the silencing effect is related to homology-dependent silencing reported in various organisms, which occurs when several copies of a gene are present in the genome and results in the specific repression of all the copies of the gene, including the endogenous one (BINGHAM 1997 ). Possibly, various fragments of R1Dm can silence IH because they mediate cosuppression triggered by the endogenous multiple copies of R1Dm that are present in the genome. If so, homologous fragments from the R1 retrotransposon of B. mori (R1Bm) should not silence IH. R1Bm and R1Dm are closely related in structure in the proteins that they encode and in insertion specificity, but they share no DNA sequence identity (JAKUBCZAK et al. 1990 ). We generated a PCR fragment, EBm, corresponding to a region in the beginning of ORF2 of R1Bm, and inserted it in IH (Fig 3). The resulting construct, IH-EBm, was introduced into the genome of the JA reactive strain by P-element-mediated transformation. Several transgenic lines were established and in situ hybridization experiments were performed on ovaries of transgenic females. Fig 4H shows that IH-EBm produces transcripts as does IH.

Silencing of a yemanuclein- chimeric transcription unit by R1Dm sequences:
To test whether the silencing properties of R1Dm sequences may act on another transcription unit, unrelated to the I factor, we designed the yema-LacZ construct. In this construct, the LacZ₉₀₀ fragment from the bacterial ß-galactosidase gene is under the control of the promoter and regulatory sequences of the yemanuclein- gene (AIT-AHMED et al. 1992 ), including the sequences of the first three exons required for the localization of the yemanuclein- transcripts in the oocyte (CAPRI et al. 1997 ). The yema-LacZ construct (Fig 3) was introduced into the genome of the JA reactive strain by P-element-mediated transformation and several transgenic lines were established. In situ hybridization experiments on ovaries using a LacZ₈₀₀ probe showed that the expression pattern of the yema-LacZ transgenes is similar to that of the endogenous yemanuclein- gene (Fig 5).

View larger version (0K): In this window In a new window Download PPT slide   Figure 5. Silencing of yemanuclein- transcription units. Typical in situ hybridization experiments on ovaries of transgenic females containing yema-LacZ (a and c) and yema-LacZ-EDm (b and d). The probe LacZ800 (a and b) detects transcripts of the yema-LacZ derivatives, and the probe yem (c and d) detects transcripts of the endogenous yemanuclein- gene (Fig 3). Arrows show some examples of RNA detection in the oocyte.

The yema-LacZ construct contains a unique XbaI site located just downstream of the ß-galactosidase sequences and upstream of the SV40 polyadenylation signal. The EDm PCR fragment from R1Dm was cloned into this XbaI site to produce the construct yema-LacZ-EDm, which was introduced into the genome of the JA reactive strain by P-element-mediated transformation. Several transgenic lines were established. In situ hybridization experiments on ovaries of transgenic females using a LacZ probe showed that, as expected, no transcripts are produced from the chimeric yema-LacZ-EDm construct (Fig 5B). Thus, as was the case for IH, silencing of the chimeric transcription unit yema-LacZ is mediated by the presence of sequences from the retrotransposon R1Dm.

I factor sequences can silence a chimeric yemanuclein- transcription unit in an inducer, not a reactive, background:
Our data suggest that, at least in the female germline, the presence of a repeated sequence within a transcription unit leads to the silencing of this unit. If so, the same transgene that undergoes silencing due to the presence of genomic repeated sequences should become fully expressed when introduced in a genome lacking these sequences. To address this question, we took advantage of the distribution of I factors in D. melanogaster. Active I factors are absent from reactive strains but exist at several copies in inducer strains, providing the opportunity to test the silencing effect of I factor sequences on the chimeric yema-LacZ transcription unit in both backgrounds.

The yema-LacZ-O1I construct was obtained by inserting the O1I PCR fragment from the ORF1 region of the I factor into the XbaI site of yema-LacZ (Fig 3). It was introduced into the genome of the JA reactive strain by P-element-mediated transformation. Two independent transgenic lines, which are reactive as the recipient JA strain, were obtained. Isogenic inducer lines were established (see MATERIALS AND METHODS) from these two lines and from two reactive transgenic lines containing the yema-LacZ construct. In situ hybridizations of RNAs to whole-mount ovaries of inducer and reactive transgenic lines were carried out using the LacZ₈₀₀ PCR fragment as probe. The results are shown in Fig 6. The transgene yema-LacZ is similarly expressed in reactive and inducer backgrounds (Fig 6, a and b), whereas the transgenes yema-LacZ-O1I containing sequences from the I factor are expressed in the reactive background (Fig 6C and Fig E) but silenced in the inducer background (Fig 6D and Fig F). These results were confirmed by using as a probe the O1I PCR fragment, which is present in yema-LacZ-O1I. This probe detects transcripts of the transgene yema-LacZ-O1I in the ovaries of the reactive, not the inducer, transgenic line (Fig 6G and Fig H). It also detects some transcripts produced constitutively by some defective I elements present in pericentromeric heterochromatin of all strains (BUCHETON et al. 1992 ). These constitutive transcripts are seen as dots in the nuclei of nurse cells (Fig 6, g–i) and, in these experiments, serve as positive controls for hybridization. Therefore, silencing of the yema-LacZ transcription unit by I factor sequences is triggered by the multiple copies of euchromatic I elements in the genome of the inducer lines.

View larger version (0K): In this window In a new window Download PPT slide   Figure 6. Silencing by I factor sequences. In situ hybridization experiments on ovaries of the JA strain (i) and of reactive (left) and inducer (right) isogenic transgenic females containing yema-LacZ (a and b) and yema-LacZ-O1I (two independent transgenic lines: one in c and d, the other in e, f, g, h, j, and k). The probes (see Fig 3) are LacZ800 (a–f), O1I (g–i), and yem (j and k). Arrows show some examples of RNA detection in the oocyte. Arrowheads show some examples of dots seen in the nurse cells nuclei with probe O1I (see text).

Silencing of a chimeric yemanuclein- transcription unit does not induce cosuppression of the endogenous yemanuclein- gene:
The yema-LacZ chimeric construct contains 2.3 kb from the 5' region of the endogenous yemanuclein- gene, including both untranscribed and transcribed sequences (CAPRI et al. 1997 ). We studied whether the silencing of the yema-LacZ transcription unit by R1Dm and I factor sequences leads to the cosilencing of the endogenous yemanuclein- gene. In situ hybridization experiments using the yem probe, specific for the endogenous yemanuclein- gene (see Fig 3), were performed on ovaries of females carrying either a yema-LacZ transgene or a yema-LacZ-EDm transgene. In both cases transcripts of the endogenous yemanuclein- gene are normally produced (Fig 5C and Fig D), although the yema-LacZ-EDm transgene is silenced (Fig 5B) and the yema-LacZ transgene is not (Fig 5A). Similarly, the endogenous yemanuclein- gene is normally expressed in the presence of yema-LacZ-O1I in both reactive and inducer backgrounds (Fig 6J and Fig K). Therefore, when the yema-LacZ-O1I transgene is silenced there is no cosuppression of the endogenous yemanuclein- gene.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Homology-dependent gene silencing triggered by retrotransposons:
Several recent independent studies have shown that I factor activity is sensitive to homology-dependent gene silencing. A reporter transgene where the chloramphenicol acetyltransferase (CAT) gene is under the control of the I factor promoter and regulatory sequences included in the 5' UTR was shown to be repressed by multiple copies of transgenes containing two or three head-to-tail tandem repeats of the I factor 5' UTR (CHABOISSIER et al. 1998 ). In addition, I factor activity in the germline of SF females was found to be partially repressed in the presence of transgenes containing coding parts of the I factor under the control of the heat-inducible hsp70 promoter (JENSEN et al. 1999A , JENSEN et al. 1999B ; MALINSKY et al. 2000 ). No particular sequence of I seems to be responsible for this repression, translation of a protein is not required, and the effect is independent of the orientation of the I sequences with respect to the hsp70 promoter. This repression is insensitive to heat shocks, but requires the presence of the hsp70 promoter and is dependent on the copy number of the transgene. Very interestingly, this repression accumulates slowly over generations, is maternally transmitted, and is maintained a few generations after transgene removal, suggesting epigenetic control (JENSEN et al. 1999A , JENSEN et al. 1999B ). Similar properties were reported for transgenes containing part of ORF1 of the I factor under the control of the actin 5C promoter (GAUTHIER et al. 2000 ). These observations indicate that the regulatory effects of the transgenes on I factor activity are related to homology-dependent gene silencing and suggest that the self-regulation of I factor activity in inducer strains might depend on such a mechanism. However, in all studies reported above, the silencing properties of the transgenes could result from their specific features (heterologous promoters or tandem repeats) and might not reflect the biological self-regulation of I factor activity.

In the present study, we demonstrate the capacity, in the female germline, of the euchromatic I elements of inducer strains to trigger homology-dependent silencing of a transgene, yema-LacZ-O1I, that contains I element sequences under the control of the yemanuclein- promoter. This transgene is not silenced in the germline of reactive females devoid of euchromatic I elements: it is expressed as is expected for a reporter construct under the control of the yemanuclein- regulatory sequences. As isogenic reactive and inducer lines were used in these experiments, the differential behavior of yema-LacZ-O1I can be attributed only to the presence/absence of euchromatic I elements. The repression observed in the inducer lines indicates that the silencing can affect sequences that are under the control of regulatory sequences other than those of the I factor. Thus, homology-mediated gene silencing triggered by the multiple euchromatic copies of I accounts at least in part for the repression of active I factors in the female germline of inducer strains.

Transcription units containing various parts of R1Dm are also repressed in the female germline. In some cases the silencing was only partial, some transgenes showing only a marked reduction of transcripts. However, our experimental approach was not quantitative and did not allow us to assess the strength of the silencing. The fact that we did not detect transcripts of certain transgenes does not mean that these transcripts are absent: they could be in amounts below the threshold of detection. Silencing phenomena described in other organisms were often found to be variable (see references in HSIEH and FIRE 2000 ). Further work, involving some quantitation of the amounts of transcripts produced by the transgenes, will be needed to address this point. Nevertheless, our data strongly suggest that the multiple endogenous copies of the R1Dm retrotransposon can trigger silencing in the female germline, although it cannot be unambiguously proven without the availability of a D. melanogaster strain devoid of R1Dm elements. Interestingly, 28S rDNA units that are interrupted by an R1Dm insertion are themselves silenced (LONG and DAWID 1979 ; LONG et al. 1981 ; JAMRICH and MILLER 1984 ), and it would be interesting to determine whether this is due to the same phenomenon.

Regulation by homology-dependent gene silencing may very well be a general feature of retrotransposons and possibly of many, if not all, transposable elements. Such a mechanism seems particularly adapted to control genome invasion by transposable elements because it does not involve a silencer or repressor brought by the element but is linked only to its repetitive nature, thereby allowing it to act on any incoming invader.

Connection with other gene-silencing mechanisms:
Future work will aim at understanding the molecular bases of homology-dependent gene silencing triggered by retrotransposons. An open question is whether such silencing occurs at the transcriptional or post-transcriptional level, or maybe both. In Caenorhabditis elegans, some mutations that affect RNAi and transgene-mediated cosuppression also lead to increased mobilization of DNA transposons (KETTING et al. 1999 ; TABARA et al. 1999 ), suggesting post-transcriptional regulation. Interestingly, in D. melanogaster, mutations in the SpindleE/homeless gene encoding an RNA helicase involved in the silencing of genomic Su(Ste) tandem repeats result in an increase of the amount of transcripts produced by some transposable elements (ARAVIN et al. 2001 ; STAPLETON et al. 2001 ). However, no increased transposition was reported in this case. On the other hand, in Arabidopsis thaliana, a protein involved in chromatin remodeling has been implicated in the regulation of retrotransposition (HIROCHIKA et al. 2000 ), suggesting transcriptional regulation. Noticeably, in Chlamydomonas reinhardtii, mobilization of a retrotransposon and of a DNA transposon are increased by mutations that affect either transcriptional (JEONG et al. 2002 ) or post-transcriptional (WU-SCHARF et al. 2000 ) silencing.

The germline silencing of the yema-LacZ-O1I transgene by multiple copies of endogenous I retrotransposons, which are themselves silenced in an inducer background, is reminiscent of the somatic silencing of the gene encoding alcohol dehydrogenase (Adh) by multiple copies (at least six) of a transgene containing the Adh-coding sequences driven by the white promoter and regulatory sequences (w-Adh; PAL-BHADRA et al. 1997 ). This silencing was shown to be transcriptional by run-on experiments and involves chromatin structure proteins of the Polycomb group. Moreover, an Adh-w transgene (w-coding sequences driven by the Adh promoter) that has no common part with the w-Adh transgenes is also silenced by the multiple copies of w-Adh transgenes, albeit only in the presence of the endogenous Adh gene (PAL-BHADRA et al. 1999 ). In this situation, the endogenous Adh gene that contains both some sequences identical to w-Adh and some sequences identical to Adh-w transgenes is believed to act as a mediator of the silencing effect between the triggering w-Adh transgenes and the target Adh-w transgene. This phenomenon is sensitive to a mutation in the piwi gene (PAL-BHADRA et al. 2002 ), a member of a family of genes that affect the RNA interference pathway. In this respect, the silencing triggered in the germline by I and R1Dm retrotransposons appears different, because silenced transgenes that contain both sequences from the yemanuclein- gene and sequences from the retrotransposon do not mediate cosuppression of the endogenous yemanuclein- gene. This suggests that, although the overall mechanisms of homology-dependent silencing are very likely to be conserved in somatic and germinal lineages, some differences presumably exist. In C. elegans, mechanisms that are based on homology-dependent silencing in the germline and in somatic tissues are closely related, but they also clearly have distinct features (DERNBURG et al. 2000 ; GRISHOK et al. 2000 ; KETTING and PLASTERK 2000 ; TIJSTERMAN et al. 2002 ).

JENSEN et al. 2002 show that multiple copies of a transgene containing sequences from the beginning of ORF2 of the I factor under the control of the hsp70 promoter can trigger silencing of a reporter transgene containing the CAT gene under the control of the I factor promoter, although both transgenes have no sequences in common. This silencing, which resembles cosuppression of w-Adh and Adh-w transgenes, might be mediated by defective heterochromatic I element sequences. By contrast, we do not observe cosuppression of the yemanuclein- gene in our studies. These differences highlight the distinct features of gene silencing involving I factors, which depend on whether they are the target or the trigger of the silencing, even though the two mechanisms presumably share a common molecular basis.

	FOOTNOTES

¹ Present address: MRC Human Genetics Unit, Crewe Rd., Edinburgh EH 2XU, United Kingdom.

	ACKNOWLEDGMENTS

We thank Ounissa Aït-Ahmed and Michelle Capri for the kind gifts of plasmids, oligonucleotides, and various tips, Pierre Couble for the gift of B. mori genomic DNA, Christine Brun for excellent technical assistance, and Alain Pélisson for very helpful discussions. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC) to A.B. S.R. was the recipient of fellowships from the the Ministère de la Recherche et de la Technologie (MRT) and from the ARC. S.C. was the recipient of fellowships from the the MRT and from the Fondation pour la Recherche Médicale.

Manuscript received November 19, 2003; Accepted for publication February 20, 2003.

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