P-Element Repression in Drosophila melanogaster by Variegating Clusters of P-lacZ-white Transgenes

Corresponding author: Stéphane Ronsseray, Laboratoire “Dynamique du Génome et Evolution,” Institut Jacques Monod, Université Paris 7, 2 place Jussieu,75251 Paris Cedex 05, France., ronsseray{at}ijm.jussieu.fr (E-mail)

Communicating editor: M. J. SIMMONS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed.

THE P-transposable element is a recent invader of natural populations of Drosophila melanogaster (KIDWELL 1983 ). Strains that possess P elements are called P strains; strains that do not are called M strains. When P males are crossed to M females, the resulting progeny exhibit a syndrome of germline abnormalities (KIDWELL et al. 1977 ) that are caused by P-element activity. This syndrome, called hybrid dysgenesis, includes a high mutation rate, chromosomal rearrangements, male recombination, and an agametic thermosensitive sterility called GD sterility (gonadal dysgenesis) that reaches 100% for some P lines, which are thus called strong P lines. The manifestation of hybrid dysgenesis appears to be repressed by various mechanisms (ENGELS 1989 ). In some populations, repression is maternally inherited—a condition called the P cytotype (ENGELS 1979 ). Of the 30–60 P elements present in one P strain genome that has been analyzed, one-third are complete P elements (BINGHAM et al. 1982 ; O'HARE and RUBIN 1983 ; O'HARE et al. 1992 ). These elements can produce an 87-kD polypeptide, the P transposase, which is required for P-element mobility (KARESS and RUBIN 1984 ; RIO et al. 1986 ). The other two-thirds of the P elements in this P strain genome are structurally incomplete; however, many of these defective P elements can still be mobilized in trans by complete P elements (ENGELS 1984 , ENGELS 1989 ) because they possess the sequences recognized by the P transposase.

The P cytotype represses transcription from the P promoter (LEMAITRE and COEN 1991 ; LEMAITRE et al. 1993 ; COEN et al. 1994 ). Although in the short term cytotype repression is maternally inherited, in the long term it is chromosomally determined by the P elements themselves (ENGELS 1979 , ENGELS 1989 ). Transformation of an M genome with P elements can induce the development of P repression over generations (ANXOLABEHERE et al. 1987 ; DANIELS et al. 1987 ; PRESTON and ENGELS 1989 ). Conversely, the removal by segregation of P elements from a P genome leads to the loss of P repression in the progeny (SVED 1987 ; RONSSERAY et al. 1993 ).

The ability of the P-element family to repress its own activity was first interpreted as resulting from the synthesis of polypeptides encoded by various P elements. ROBERTSON and ENGELS 1989 and MISRA and RIO 1990 have shown that an artificially modified P element that encodes a 66-kD truncated transposase can partially repress P-element transposition. In addition, P elements with a specific internal deletion, called KP elements, are present in high copy number in natural populations and appear to possess repression ability (BLACK et al. 1987 ; RASMUSSON et al. 1993 ). ANDREWS and GLOOR 1995 have shown that single insertions of a KP element construct driven by the actin-5 promoter have a strong ability to repress dysgenic sterility in a position-independent manner. However, insertions of a similar construct in which the KP sequence that encodes a leucine zipper motif has been modified do not repress sterility. Thus, at least in some cases, P repression appears to involve the production of P-encoded repressor polypeptides.

However, P repression may not depend exclusively on the production of P-encoded repressor polypeptides (ROCHE and RIO 1998 ; RONSSERAY et al. 1998 ). In particular, we have shown that P elements at the X chromosome telomere (cytological site 1A) regulate genome-wide P-element activity even though they are expressed at low levels. Indeed, although the P elements at 1A are responsible for a detectable level of P tranposase (RONSSERAY et al. 1996 ), RT-PCR and Northern blot analysis indicate that they are expressed only weakly (ROCHE et al. 1995 ; MARIN et al. 2000 ). These regulatory P elements are flanked by subtelomeric noncoding sequences (RONSSERAY et al. 1996 ) called telomeric-associated sequences (TAS; KARPEN and SPRADLING 1992 ), which exhibit some of the properties of heterochromatin, including the ability to silence transgenes inserted within them (LEVIS et al. 1985 ; WALLRATH and ELGIN 1995 ; CRYDERMAN et al. 1999 ; GOLUBOVSKY et al. 2001 ). In addition, P-lacZ insertions at cytological site 1A and a P-white-rosy insertion at an autosomal telomere (100F), also inserted in TAS, have been shown to prevent germline expression of a euchromatic P-lacZ insertion, a phenomenon termed trans-silencing (ROCHE and RIO 1998 ). These telomeric P transgenes also stimulate the regulatory properties of other P elements in a P strain genome (RONSSERAY et al. 1998 ). However, the telomeric P-transgene insertions do not by themselves significantly repress the dysgenic sterility induced by a strong P strain (RONSSERAY et al. 1998 ).

The repression ability of the telomeric P elements at cytological site 1A is sensitive to the dose of HP1 (RONSSERAY et al. 1996 ), a nonhistone heterochromatin protein that binds mostly to centromeres and telomeres (JAMES and ELGIN 1986 ; JAMES et al. 1989 ; WUSTMANN et al. 1989 ; FANTI et al. 1998 ); heterozygosity for a null allele of Su(var)205, the gene that encodes this protein, strongly impairs the repression ability of telomeric P elements (RONSSERAY et al. 1996 , RONSSERAY et al. 1997 ). DORER and HENIKOFF 1994 have found that tandem repeats of P-lacZ-white transgenes, inserted in euchromatin, show a variegating phenotype for the white marker and that the level of variegation is positively correlated with the number of transgenes. In addition, this variegation is suppressed by a Su(var)205 mutation. Immunostaining on polytene chromosomes indicates that a cluster with a high number of P-lacZ-white transgenes creates a new HP1 binding site despite its location in euchromatin (FANTI et al. 1998 ). Because the P-lac-w clusters appear to be heterochromatinized, we hypothesized that they might show some ability to repress P-element activity similar to that of P-lacZ elements inserted in subtelomeric heterochromatin. In this article, we show that some P-lac-w transgene clusters are able to repress the dysgenic sterility induced by a strong P strain and that they repress the germline expression in trans of P-lacZ elements through a maternal effect.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
Canton^y is a typical M strain (KIDWELL et al. 1977 ) containing no P elements and marked with a spontaneous mutation of yellow.

Harwich-2 is a strong P strain. The subline used here shows >80 P labels by in situ hybridization on polytene chromosomes. It carries an unidentified autosomal recessive marker (sepia-colored eye), which appeared spontaneously in the stock.

Lk-P(1A): This line carries two autonomous P elements inserted in TAS at the X chromosome telomere (cytological site 1A; RONSSERAY et al. 1996 ). These elements, derived from a natural population from Azerbaïjan (BIEMONT et al. 1990 ), were isolated by genetic recombination in a background devoid of other P elements (RONSSERAY et al. 1991 ); this strain has a strong ability to repress P activity in the germline and the repression ability is maternally inherited (P cytotype).

BQ16/Cy, BC69/Cy, and ABOO/Cy: These three lines carry a P-lacZ-rosy enhancer-trap insertion (P[lac, ry⁺]A, renamed P{A92} in Flybase) at different sites on the second chromosome. The lacZ coding sequence is fused in frame with the first 587 bp of P, a sequence that includes exon 0 and part of exon 1 of the P element. ß-Galactosidase staining is mostly nuclear since the 5' part of the P element carries a nuclear localization signal. These insertions express ß-galactosidase activity in the germline tissues of the ovaries and testes (J. L. COUDERC and F. A. LASKI, personal communication). In addition, ABOO is expressed in the somatic follicle cells. The genes adjacent to these insertions are still unidentified except for the BC69 insertion, which is near the gene vasa.

R3-29: This P-lacZ insertion, located in 1A-B on the X chromosome, is expressed only in late stage nurse cells. The genes adjacent to this insertion are unidentified.

P-Co-1: This line carries a homozygous viable insertion of pCo (P-white-otu-lacZ; see below) on the third chromosome (87A-B, according to an inverse PCR cloning procedure; DDBL/EMBL/GenBank accession no. AC007889). ß-Galactosidase expression in this transgene is driven by the otu promoter and is therefore strongly detected in both nurse cells and the mature oocyte. The staining is cytoplasmic because the ß-galactosidase encoded by this construct does not possess a nuclear localization signal.

P-1039: cn¹ ; ry⁵⁰⁶.This line, provided by Allan Spradling, carries a P-lacZ-ry transgene inserted at 60B. This transgene is strongly expressed in the female germline (nurse and follicle cells).

P-1152: P[ry^+t7.2 = lArB]A171.1F1; ry⁵⁰⁶ (synonym WG-1152). This line, from Walter Gehring, carries a P-lacZ-ry-adh construct at cytological site 1A. It is inserted in TAS (ROCHE and RIO 1998 ). No LacZ expression is detected in the female germline, even after overnight staining.

P-lac-w clusters: Seven lines with different numbers of P-lacZ-white elements (P[lacZ^P/I.W w^+mC ampR ori = lacw]; BIER et al. 1989 ) located at cytological site 50C on the second chromosome (DORER and HENIKOFF 1994 , DORER and HENIKOFF 1997 ). The P-lacZ-white construct is 10,691 bp long, contains the P-lacZ translational fusion at position 587 of the P sequence, and is marked by the mini-white gene (Fig 1A). The white gene, used initially as a transformation marker, is also used as a reporter for the level of repression of the transgenes. Variegating clusters of transgenes were first generated at cytological site 92E (DORER and HENIKOFF 1994 ). Mobilization of a tandem duplication at this site, by the P-element transposase, led to the recovery of a tandem transgene duplication at cytological position 50C in the middle of the euchromatic region of the right arm of a nonrearranged second chromosome (line 1A-6; DORER and HENIKOFF 1994 ). Flies hemizygous for the 50C duplication showed wild-type red eye pigmentation with no mottling at any temperature. Secondary mobilization of the tandem duplication step with transposase activity (Fig 1C) led to the excision of one of the two elements, thereby creating a strain with a single insertion at 50C (line 6-2, hemizygous with orange eye pigmentation). In the same mobilization experiment, increased transgene copy number at 50C was also recovered (four copies, line 6-4). At 20°, the 6-4 hemizygous flies showed red eye pigmentation with some white spots. Further mobilization led to strains with six or seven transgene copies at 50C (lines DX1 and BX2, respectively). At 20°, these strains also showed red pigmentation at 20° with unpigmented spots. Finally, X-ray mutagenesis was performed on BX2 males to induce chromosomal rearrangements (DORER and HENIKOFF 1997 ). The C-2 and T-1 lines thus derived showed large unpigmented sectors in otherwise red eyes, indicating very strong enhancement of the variegating repression on the white gene in the P-lacZ-white transgenes. C-2 has a rearrangement involving the second chromosome (2R arm), whereas T-1 has complex chromosomal rearrangements, including translocations between the second and the third chromosomes. The single insertion at 50C (6-2) is fully fertile and can be maintained in the homozygous state. In contrast, all clusters of insertions are homozygous lethal or sterile and must be balanced with a Cy-marked second chromosome. After overnight staining, LacZ expression is detected in the follicle cells of all lines, presumably because of a position effect at 50C (see the RESULTS and Fig 3L). No staining is detected in nurse cells or in oocytes. The main properties of these lines are summarized in Fig 1B and Fig C.

View larger version (26K): In this window In a new window Download PPT slide   Figure 1. Nomenclature, structure, and origin of the transgene clusters. (A) The autonomous P element is shown as well as the P-lac-w transgene present in the clusters. The arrowheads indicate the 31-bp inverted terminal repeats of the P element. The HindIII restriction sites used in Southern blot analysis are indicated as H. (B) The nomenclature and description of the lines is given. (C) The relationships among the different lines is given along with the agents used to modify them.

View larger version (18K): In this window In a new window Download PPT slide   Figure 2. Southern blot analysis of transgene clusters. HindIII digestion of genomic DNA. The blot was probed with a 650-bp PCR-amplified fragment corresponding to the BclI fragment located 5' to white (BOIVIN and DURA 1998 ).

View larger version (126K): In this window In a new window Download PPT slide   Figure 3. (A–K) Repression assay for P-lacZ in BQ16. The BQ16 insertion expresses ß-galactosidase in the ovarian germline tissues (nurse cells and mature oocyte). Each picture shows the staining of ovaries of G1 females from the cross of tested females with BQ16 males, reared at 20°. Tested females: (A) Cantony, (B) Lk-P(1A), (C) P-1152, (D) P-605, (E) 6-2, (F) 1A-6, (G) 6-4, (H) DX1, (I) BX2, (J) C-2, (K) T-1. Staining reactions were performed overnight in the same experiment to allow a visual comparison. (L) Overnight staining of ovaries from the T-1 line performed separately. n.c., nurse cells; oo., oocyte; f.c., follicle cells.

P-605: y w; . This line carries an insertion of a P-lacZ-white enhancer trap at 39E on chromosome 2.

w; T-1/Cy Roi; P-Co-1: This line carries both the T-1 cluster on the second chromosome and the P-Co-1 insertion on the third chromosome. The balancer of the second chromosome carries two dominant markers, Curly and Roi.

w^m4; Su(var)(205)⁵/Cy; +/+: This stock carries a Su(var)205 mutant allele, which encodes only the first 10 amino acids of the HP1 protein (EISSENBERG et al. 1992 ). This allele strongly impairs the repression ability of Lk-P(1A) (RONSSERAY et al. 1996 , RONSSERAY et al. 1997 ).

Making of the pCo plasmid:
The otu promoter was amplified by PCR from the pCOG plasmid (ROBINSON and COOLEY 1997 ) with oligonucleotide primers containing additional restriction sites at the 5' end (EcoRI and BamHI, underlined) for subcloning purposes. Primers used were 5'-CGGAATTCATAGTCGTTGCG-3' and 5'-CGGGATCCGTTAACAATTAAATTAAC-3'. The PCR-amplified product was then digested with EcoRI and BamHI and cloned into pCaSpeR-AUG-ß-gal (THUMMEL et al. 1988 ) digested with the same enzymes. This new construct was named pCo and will be figured in the RESULTS section. Transgenic lines were obtained as previously described in BOIVIN and DURA 1998 .

Southern blot analysis:
Genomic DNA was extracted as described previously (BOIVIN and DURA 1998 ). A total of 25 µl of the DNA preparation was digested to completion with HindIII. The DNA was then size fractionated on a 1% agarose gel and transferred to a reinforced cellulose nitrate membrane. The blot was hybridized with the 650-bp BclI fragment from the 5' region of the white locus, [³²P-dCTP]-labeled by random priming [Boehringer Mannheim (Indianapolis) High Prime kit] in 6x SSC, 5x Denhardt's solution, 0.5% SDS at 65° for 12 hr, and then washed three times for 10 min with 2x SSC, 0.1% SDS at 65°. The autoradiogram was visualized on RX Fuji medical X-ray film.

Gonadal dysgenesis assay:
The ability of lines to repress the occurrence of GD sterility was measured by the A* assay (KIDWELL et al. 1977 ). Females of the tested line were crossed with strong P males (Harwich-2). For each testcross, 3–10 pairs were mated en masse and placed at 29°. Parents were discarded after 3 days of egg laying. Approximately 2 days after the onset of eclosion, G₁ progeny were collected and allowed to mature for 2 days at room temperature. A total of 25 to 50 G₁ females were then taken at random for dissection. Dissected ovaries were scored as unilaterally dysgenic (S1 type) or bilaterally dysgenic (S0 type; SCHAEFFER et al. 1979 ). The frequency of gonadal dysgenesis was calculated as %GD = %S0 + ¹/₂%S1 and will be referred to as percentage of GD A* (%GD A*). The M cytotype, which allows P elements to be active, results in a high percentage of GD A*, whereas the P cytotype, which represses P-element activity, results in a low percentage of GD A* (<5%). An intermediate percentage indicates incomplete repression.

Repression of P-lacZ expression in ovaries:
G₁ females derived from a cross between individuals from a tested line and individuals from lines bearing a P-lacZ transgene were examined by staining for their ability to repress P-lacZ expression in the ovaries. Staining of ovaries to detect lacZ expression was performed as described in LEMAITRE et al. 1993 . High lacZ activity indicates an absence of P repression and low activity indicates strong P repression (LEMAITRE et al. 1993 ).

Assay of the Su(var)205 mutant allele effect on the ability of T-1 to repress a transgene in the germline:
Females with the genotype w; T-1/Cy Roi; P-Co-1 were crossed with w^m4; Su(var)205⁵/Cy; +/+ males at 25°. G₁ females bearing both the T-1 and the P-Co-1 transgenes (phenotype [Roi⁺]) as well as the Su(var)205⁵ mutant allele (phenotype [Cy⁺]) were compared to their sisters [Cy Roi⁺] for lacZ expression in ovaries. As a positive control of lacZ expression, G₁ females that inherited the P-Co-1 insertion but not the cluster (phenotype [Cy Roi]) were also stained.

Statistical analysis:
The repression abilities as measured by GD sterility percentages were compared using the nonparametric Mann-Whitney test performed on A* assay replicates.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular comparison of the clusters:
X-ray irradiation, performed on the line BX2, led to the recovery of the T-1 line (DORER and HENIKOFF 1997 ). This line carries chromosomal rearrangements that are thought to be responsible for the enhancement of the variegated repression of the white marker. We compared the structure of the clusters in the BX2 and T-1 lines to determine whether X-ray irradiation had also altered the structure of the cluster. Genomic DNA was digested by HindIII, which cuts at position 40 inside the 5' P-element sequence and at positions 10,272 and 10,453 in the pUC sequence of the P-lac-w transgene. The Southern blot was probed with a 650-bp fragment from the 5' side of the white gene. This is expected to hybridize to a 10.2-kb internal fragment from an intact P-lac-w transgene. Fig 2 shows that 6-2 and 6-4 DNAs produce the full-size HindIII fragment. However, 6-4 also produces a second shorter fragment corresponding to a deleted transgene. This deletion is known to remove most of the pUC sequences and the 3' polylinker from one P-lac-w element, but the lacZ and white sequences appear intact (D. DORER, personal communication). BX2 shows the same pattern as 6-4, indicating the presence of both intact transgenes and of transgenes deleted in the same way. Finally, T-1 shows the same pattern as BX2. Thus, at this level of resolution, this indicates that the X rays used on BX2 to induce the chromosomal rearrangements present in T-1 did not alter the internal structure of the transgene cluster in T-1.

P repression capacities of the clusters:
The ability of the lines to repress P-element activity was tested with four different assays.

(a) Repression of GD A* sterility (Table 1, left): G₁ females reared at 29° and generated from the cross of M females (Canton^y) with strong P males (Harwich-2) are sterile due to atrophy of the gonads (100% gonadal dysgenesis). Conversely, females with the P cytotype [Harwich-2 or Lk-P(1A)] crossed with strong P males produce G₁ females with trivial percentages of GD sterility (0.3 and 1.1%, respectively) due to the repressive component transmitted by the P females. Lines with a single P-lacZ do not significantly repress GD sterility (see P-1152, P-605, and 6-2). Among lines carrying tandem arrays of P-lac-w transgenes, only one represses GD sterility. The exception is the T-1 line, which partially but significantly represses the occurrence of dysgenic sterility (74.3% GD). The comparison of the T-1 repression ability with that of the M control (Canton^y) is highly significant (P < 0.01) as judged by the Mann-Whitney test. In addition, for some of the GD assay replicate tests (16 out of 20) performed with T-1, the G₁ females bearing the cluster [Cy⁺] and their siblings lacking the cluster [Cy] were dissected separately. Similar percentages of GD sterility were found for the two kinds of progeny: [Cy⁺], m = 77.2%, s = 15.5, n = 16; [Cy], m = 79.3%, s = 16.6, n = 16. This shows that G₁ females that did not inherit the T-1 cluster nonetheless inherited the capacity to repress GD.

(b) Combination effect between tested lines and a P-lacZ telomeric insertion: The combination of a telomeric transgene with regulatory P elements results in a combination effect, i.e., an enhancement of the repression ability of the regulatory P elements (RONSSERAY et al. 1998 ). We tested if the combination of such a telomeric transgene with a transgene cluster leads to enhancement of the ability of the cluster to repress GD sterility. We crossed P-1152 females, which carry a P-lacZ element at the telomere of the X chromosome, with males of the lines under test and measured the repression abilities of the G₁ females by crossing sets of these females with P-strain males (Harwich-2) at 29° (Table 1, right). As expected, the cross of P-1152 females with M males (Canton^y) produced G₁ females with no repression capacity (99.6% GD). As shown previously (RONSSERAY et al. 1998 ), crossing these females with Lk-P(1A) males produces females with strong repression ability (33.1% GD). However, no significant enhancement of the repression ability of G₁ females bearing the P-1152 telomeric transgene with a single P-lac-w (P-605, 6-2) or with two P-lac-w copies (1A-6) was observed. By contrast, all the other clusters with a higher number of transgenes showed enhanced repression of GD sterility. The maximum was obtained with T-1 (21.0% GD).

Taken together, these results show that a cluster (T-1) can elicit a significant repression of dysgenic sterility by itself and that the combination of a cluster (four copies or more) with a telomeric P-lacZ leads to enhanced levels of repression. In addition, strong repression (21.0% GD) can be reached by combining T-1 with P-1152.

(c) Repression of a P-lacZ element in the germline: The BQ16 P-lacZ insertion was used in this assay. In an M context, this insertion is strongly expressed in the nurse cells and in the oocyte (Canton^y, Fig 3A). G₁ females from the crosses between Lk-P(1A) females and BQ16 males strongly repressed lacZ activity in the germline (Fig 3B). However, weak staining in follicle cells (due to BQ16) is still detected. Such a restriction of Lk-P(1A) repression ability to germline tissue has previously been shown (RONSSERAY et al. 1991 ; LEMAITRE et al. 1993 ). Females carrying the P-1152 transgene also strongly repressed BQ16 expression (Fig 3C). This result is consistent with that of ROCHE and RIO 1998 and MARIN et al. 2000 in which P-1152 repressed a P-lacZ transgene in the germline. Among the other lines tested, T-1 completely repressed BQ16 expression in the germline (nurse cells; Fig 3K) whereas other clusters did not (Fig 3, D–J), except for slight repression with C-2. Therefore, like P-1152, T-1 shows the ability to silence, in trans, a homologous transgene specifically in the germline.

(d) Repression of various P-lacZ insertions: We tested whether germline silencing by the T-1 line is restricted to the BQ16 insertion or whether it can be extended to other transgenes. We tested four other P-lacZ insertions that are expressed in the germline. ABOO, BC69, and P-1039 are located on the second chromosome and R3-29 is located on the X chromosome. The expression pattern of each insertion was determined in the ovaries of female progeny from crosses between males from lines bearing these insertions and Canton^y females (Fig 4A, Fig C, Fig E, and Fig G). In parallel, the same males were crossed with T-1 females (Fig 4B, Fig D, Fig F, and Fig H). In each case, the T-1 cluster completely repressed the activity of the P-lacZ in the nurse cells. T-1 did not show any repression of ABOO and P-1039 expression in somatic follicle cells (Fig 4D and Fig H).

View larger version (96K): In this window In a new window Download PPT slide   Figure 4. Ability of T-1 to repress several transgenes at different sites. Four different P-lacZ transgenes expressed in the female germline were tested as targets. (A–B) BC69, (C–D) ABOO, (E–F) R3-29, (G–H) P-1039. In each case, P-lacZ males were crossed with Cantony females at 20° (left) or T-1 females (right). For each line tested, staining reactions were performed overnight in the same experiment to allow a visual comparison.

Finally, we used a different construct as a target to test whether the proximity of the promoter driving LacZ expression and P sequences is a necessary condition for silencing to occur. The P-Co-1 line contains a transgene named P-Co (see MATERIALS AND METHODS and Fig 5E) in which lacZ is not fused in frame with the 5' P sequence but is driven by the otu promoter. In this construct, the otu promoter is located at a central position between white and lacZ, i.e., >3 kb away from the P sequences. Because of the otu promoter, LacZ is strongly expressed in the germline (Fig 5A). The transgene is completely repressed by P-1152 and T-1 (Fig 5B and Fig C). However, P-Co-1 is not repressed by the full-sized P elements of Lk-P(1A) (Fig 5D).

View larger version (113K): In this window In a new window Download PPT slide   Figure 5. Ability to repress a transgene driven by a heterologous promoter. The P-Co-1 line containing the P-Co transgene, in which lacZ is under the control of the otu promoter, was tested as a target. Each picture shows the staining of ovaries of G1 females from the cross of tested females with P-Co-1 males, reared at 25°. (A) Cantony, (B) P-1152, (C) T-1, (D) Lk-P(1A). Staining reactions were performed overnight in the same experiment. (E) Structure of the P-Co transgene.

Taken together, these results show that the T-1 cluster located at 50C on the second chromosome can repress germline expression of another P-lacZ, located either on the same chromosome at a different site (BC69 in 35C) or on a different chromosome (R3-29, on the X chromosome; P-Co-1 on chromosome 3). Therefore, the T-1 cluster causes homologous transgene silencing on a target located at a nonallelic position. In fact, silencing was found with all the targets tested, regardless of their insertion site (and therefore the neighboring enhancer) and regardless of their structure with respect to the position of the promoter driving LacZ expression. This last point suggests that this silencing can result not only from the P-element sequences shared by the silencer and the target, but also from the shared lacZ sequences. Such a mechanism is consistent with the lack of significant repression of P-Co-1 by Lk-P(1A) P elements, which have no lacZ sequences.

Maternal effect of the trans-silencing:
The regulatory properties of the naturally occurring autonomous P elements at 1A show a strong maternal effect for both the repression of dysgenic sterility (RONSSERAY et al. 1991 , RONSSERAY et al. 1993 ) and the repression of a P-lacZ element (LEMAITRE et al. 1993 ). We tested whether the repression abilities of a telomeric P-lacZ insertion (P-1152) and of the T-1 cluster also show a maternal effect. For these two lines, the two reciprocal crosses were performed with BQ16 at 20° and G₁ females were stained overnight for LacZ activity. Fig 6 shows that the repression ability of both P-1152 and T-1 involves a strong maternal effect. Repression is detected only when the P-1152 or T-1 transgenes are maternally inherited. The trans-silencing properties of T-1 therefore present strong similarities with that of the telomeric P-reporter P-1152.

View larger version (106K): In this window In a new window Download PPT slide   Figure 6. Maternal effect assay on trans-silencing. Pictures show the overnight staining of ovaries of G1 females reared at 20°. (A) P-1152 females x BQ16 males; (B) BQ16 females x P-1152 males; (C) T-1 females x BQ16 males; (D) BQ16 females x T-1 males. For each line tested (P-1152 and T-1), staining reactions were performed overnight in the same experiment.

Assay of the Su(var)205 mutant allele effect on the T-1 repressive properties:
We have tested whether the trans-silencing induced by T-1 is affected by the dose of Su(var)205 that encodes HP1. Indeed, it is known that the variegating phenotype of the clusters is suppressed by reducing the dose of HP1 (DORER and HENIKOFF 1994 ). In addition, HP1 was shown to bind the clusters in 50C with an intensity correlated to the number of transgenes (FANTI et al. 1998 ). Ovaries from females having the P-Co-1 transgene and the T-1 cluster and from sibling females having the same chromosomal complement but heterozygous Su(var)205⁵/Su(var)205⁺ were compared after overnight staining for lacZ. The two kinds of females showed a strong repression in P-Co-1 expression, similar to that observed in Fig 5C, whereas the control G₁ females having the P-Co-1 transgene without the T-1 cluster showed a strong staining similar to that observed in Fig 5A. Therefore, the presence of the Su(var)205⁵ mutated allele did not impair the repression ability of T-1.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Repression of P-induced dysgenic sterility in the absence of a repressor-encoding P element:
The P-element family is able to establish the conditions for repression of its own activity. Transformation of an M genome with P elements can induce the development of P repression over generations (ANXOLABEHERE et al. 1987 ; DANIELS et al. 1987 ; PRESTON and ENGELS 1989 ). The ability of a given P element to establish the P cytotype depends on its structure. Various studies have allowed the identification and characterization of some regulatory P elements. These include autonomous P elements (RONSSERAY et al. 1991 , RONSSERAY et al. 1996 ; O'HARE et al. 1992 ), copies lacking the last intron (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; GLOOR et al. 1993 ; MISRA et al. 1993 ), and copies with a central deletion, termed KP elements (BLACK et al. 1987 ; HIGUET et al. 1992 ; RASMUSSON et al. 1993 ; ANDREWS and GLOOR 1995 ). In each case, the P copy is able to encode a repressor polypeptide that is thought to repress P-element activity by transcriptional repression (LEMAITRE and COEN 1991 ; LEMAITRE et al. 1993 ) or by protein-protein interactions (ANDREWS and GLOOR 1995 ).

It appears also that the genomic location of the P element is crucial with regard to its repression capacity. Two copies of autonomous P elements at the X chromosome telomere exhibit strong repression ability, as strong as that of a P strain carrying 50 P elements scattered throughout the genome (RONSSERAY et al. 1996 ). In addition, when located at this site, a P-lacZ transgene, which almost certainly does not encode a repressor polypeptide, nonetheless has some repression ability (ROCHE and RIO 1998 ; RONSSERAY et al. 1998 ): it is able to repress in trans a P-lacZ at another site in the genome, and it does this specifically in the germline. Only telomeric locations appear to confer such repression ability to P-reporter transgenes. At telomeric sites, P insertions are inserted in subtelomeric heterochromatin (KARPEN and SPRADLING 1992 ; RONSSERAY et al. 1996 ; ROCHE and RIO 1998 ; CRYDERMAN et al. 1999 ) and no LacZ expression can be detected in the germline even after overnight staining (data not shown). The repression capacities of telomeric P insertions have thus been proposed to result from their ability to interact at the chromatin level with euchromatic targets (ROCHE and RIO 1998 ; RONSSERAY et al. 1998 ). Alternatively, it can be proposed that they produce aberrant RNAs that lead to RNA interference. However, among the three telomeric transgenes that cause a trans-silencing effect, none can repress significantly, by themselves, the dysgenic sterility induced by a strong P strain (RONSSERAY et al. 1998 ).

The clusters of P-lac-w transgenes analyzed here were suspected to share some properties with telomeric transgenes because they are also heterochromatinized due to their repeated structure (DORER and HENIKOFF 1994 , DORER and HENIKOFF 1997 ; FANTI et al. 1998 ). The present study shows that one of them, T-1, has a significant ability to repress the GD sterility induced by a strong P strain (25% of the ovaries develop in the GD A* assay). Furthermore, when combined with a telomeric P-lacZ (P-1152), clusters with four copies or more have significant GD repression abilities. In the case of T-1, the repression ability is strongly enhanced (79% of developed ovaries). One case of P-induced GD sterility repression, without the production of any P repressor, was described previously (RASMUSSON et al. 1993 ). A 0.5-kb P element called SP, which encodes only the first 14 amino acids of the transposase, was found to have the ability to repress the GD sterility induced by a weak P strain. Our results extend this result and show that the capacity to repress the dysgenic sterility induced by a strong P strain can be established in the absence of a P-encoded polypeptide repressor by, for example, combining a P transgene in subtelomeric heterochromatin and the T-1 cluster.

Comparison between BX2 and T-1:
Both the T-1 and BX2 clusters have repressive properties in the occurrence of dysgenic sterility when combined with the telomeric P-lacZ transgene in the P-1152 line. However, T-1 has stronger abilities than BX2. In particular, T-1 induces strong trans-silencing of a P-lacZ transgene in the germline, whereas BX2 does not. The T-1 line was derived upon X-ray treatment of the BX2 line (DORER and HENIKOFF 1997 ). What accounts for the different properties of these two lines? Southern blot analysis showed that the cluster itself is not rearranged in the T-1 line when compared to the BX2 line (Fig 2). We also carried out in situ hybridizations on polytene chromosomes of the T-1 line with a P-element probe. Chromosomal rearrangements, including translocations, led to uninterpretable cytology, but only one P-hybridizing site was found (data not shown). This indicates that the cluster is still the only P sequence in the genome; it has not been divided into pieces and scattered throughout the genome. In addition, the site appears to be located in a median section of a chromosome segment: it is not at a neotelomeric position or close to a centromere.

If we compare the variegating eye color phenotype of the T-1 and BX2 lines in flies, the T-1 cluster appears to be much more heterochromatinized than the BX2 cluster. Although the in situ hybridization label indicates that the T-1 cluster is not close to a centromere, it remains possible that the X-ray mutagenesis performed on BX2 flies resulted in the translocation of a heterochromatic block in the vicinity of the cluster of transgenes. Precedence for this exists in the case of a translocation resembling that which occurred at the brown^D allele, which was generated upon insertion of a large block (1 Mb) of centric heterochromatin inside the brown gene on the second chromosome (SLATIS 1955 ). In the T-1 line, a translocation of heterochromatin near the cluster could mimic the situation that occurs when a transgene is in subtelomeric heterochromatin, thereby conferring an increased P repression ability.

What is the mechanism of P repression by transgene clusters?
Does the repression ability of T-1 involve a diffusible product of the cluster, an altered structure of the chromatin, or both? Under the diffusible product hypothesis, this product is probably RNA rather than protein since the T-1 cluster has no detectable expression in the nurse cells (Fig 3L). RNA interference may occur in Drosophila (KENNERDELL and CARTHEW 1998 ; ARAVIN et al. 2001 ) and has already been proposed to play a role in transposable element regulation (JENSEN et al. 1999A , JENSEN et al. 1999B ; ARAVIN et al. 2001 ). Regarding P repression, SIMMONS et al. 1996 have shown that expression of antisense RNAs can lead to partial P repression. In addition, cosuppression has also been shown to exist in Drosophila both at the transcriptional and post-transcriptional levels (PAL-BHADRA et al. 1997 , PAL-BHADRA et al. 1999 ; U. BHADRA, M. PAL-BHADRA and J. BIRCHLER, personal communication). In the experiments performed to assay the ability of T-1 to repress GD sterility, the G₁ females bearing the cluster and their sisters that carry the Cy balancer chromosome (and thus no P transgene) produced similar percentages of GD sterility, suggesting that a diffusible product is deposited in the oocyte and that this product is responsible for the observed repression of P activity in the offspring. This product could also explain the maternal effect of the trans-silencing induced by T-1.

However, it remains possible that a change in the chromatin at the level of the cluster contributes to the system. In such a model, telomeric sequences and the cluster could pair with homologous sequences in the genome and inactivate them in trans, either by trans-heterochromatinization or by relocalization of the sequences to another compartment of the nucleus. Alternatively, altered chromatin structure could be responsible for the production of aberrant RNAs with the ability to induce RNA interference (VOINNET et al. 1998 ). A good candidate for the induction of a change in the state of the chromatin could be HP1 since it has been shown to bind P-lac-w clusters at 50C (FANTI et al. 1998 ). Surprisingly, we show here that the ability of T-1 to induce trans-silencing is not affected by the dose of HP1, suggesting that the normal dose of HP1 is not a necessary condition. Furthermore, HP1 binding on the clusters of P-lac-w transgenes is not a sufficient condition because clusters with two to seven copies, but with no rearrangements, are strong binding sites for HP1 on polytene chromosomes (FANTI et al. 1998 ), although they do not induce trans-silencing. An alternative explanation is that an effect of a Su(var)205 mutated allele on trans-silencing requires maternal transmission of the mutated allele. Here, the Su(var)205⁵ allele was introduced paternally because it is not possible to introduce it maternally since this would require recombination between the rearranged T-1 chromosome and a nonrearranged chromosome 2, carrying the Su(var)205 mutated allele.

Trans-silencing effect: toward a general homology-dependent phenomenon:
We have previously shown that the ability of a telomeric P insertion to repress the expression of a P transgene in the germline requires some length of homology between the two P sequences (MARIN et al. 2000 ). Indeed, a naturally occurring, defective P element at 1A [namely, NA-P(1A)], deleted for the first 871 bp at the 5' side, is unable to repress a classical P-lacZ construct. In that case, only 233 bp are present both in the deleted P element and the P transgene. However, NA-P(1A) is able to repress a particular P-lacZ construct (namely, PLH3). In this case, 1.8 kb are present both in the deleted P element and the P transgene. In the present work, the telomeric full-sized P elements of the Lk-P(1A) line do not repress P-Co-1 (Fig 5). Thus, the 800 bp of the P sequence (587 bp at the 5' side and 233 bp at the 3' side), present both in Lk-P(1A) and P-Co-1, are not sufficient to induce silencing. However, the T-1 cluster and the P-1152 telomeric P-lacZ insertion strongly repress the P-Co-1 insertion. These results strongly suggest that the capacity of T-1 or P-1152 lines to suppress P-Co-1 expression results not only from the 800 bp of P-element sequences shared by the silencer and the target, but also from the shared lacZ sequence (3 kb), suggesting that this kind of suppression can be induced by a non-P-element sequence. We are currently investigating whether a hobo-lacZ transgene, expressed in the germline, can be repressed by a telomeric P-lacZ, due simply to the lacZ sequence homology. Trans-silencing induced by telomeric sequences could in fact represent a general phenomenon involved in some cellular functions of the germline. Such a suppression might have been adopted by the P-element family after its recent invasion of natural D. melanogaster populations, allowing its own regulation.

	ACKNOWLEDGMENTS

We thank Doug Dorer, Manika Pal-Bhadra, Utpal Bhadra, and James Birchler for personal communications. We thank J. L. Couderc for providing P-lacZ lines. We thank A. M. Pret and S. Charlat for their help in the preparation of the manuscript. We thank the communicating editor M. J. Simmons and an anonymous reviewer for their valuable comments and suggestions on the manuscript. S.R. thanks Jocelyne Besombe for helpful suggestions. We thank the Bloomington and Umea Stock centers for providing stocks and FlyBase for helpful informations. This work was supported by the Centre National de la Recherche Scientifique (UMR 7592), by the program Génome, by the Association pour le Recherche sur le Cancer, and by the Universités Paris 6-Pierre et Marie Curie and Paris 7-Denis Diderot (Institut Jacques Monod-UMR7592, Dynamique du Génome et Evolution). This work was carried out in compliance with the current laws governing genetic experimentation in France and in the United States.

Manuscript received December 12, 2000; Accepted for publication September 24, 2001.

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