Repression of Hybrid Dysgenesis in Drosophila melanogaster by Combinations of Telomeric P-Element Reporters and Naturally Occurring P Elements

Corresponding author: Stéphane Ronsseray, Département 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 melanogaster, hybrid dysgenesis occurs in the germline of flies produced by crosses between females lacking P elements and males carrying 25–55 P elements. We have previously shown that a complete maternally inherited repression of P transposition in the germline (P cytotype) can be elicited by only two autonomous P elements located at the X chromosome telomere (cytological site 1A). We have tested whether P transgenes at 1A, unable to code for a P-repressor, may contribute to the repression of P elements. Females carrying a P-lacZ transgene at 1A ["P-lacZ(1A)"], crossed with P males, do not repress dysgenic sterility in their progeny. However, these P-lacZ(1A) insertions, maternally or paternally inherited, contribute to P-element repression when they are combined with other regulatory P elements. This combination effect is not seen when the P-lacZ transgene is located in pericentromeric heterochromatin or in euchromatin; however a P-w,ry transgene located at the 3R chromosome telomere exhibits the combination effect. The combination effect with the P-lacZ(1A) transgene is impaired by a mutant Su(var)205 allele known to impair the repression ability of the autonomous P elements at 1A. We hypothesized that the combination effect is due to modification of the chromatin structure or nuclear location of genomic P elements.

THE P-transposable element has invaded natural Drosophila melanogaster populations over the last five decades (KIDWELL 1983 ). After a transient period of dysgenesis, various mechanisms of repression have developed that are, depending on the population, maternally inherited (P cytotype) (ENGELS 1979 , ENGELS 1989 ) or biparentally transmitted (P susceptibility) (BLACK et al. 1987 ; SIMMONS et al. 1990 ). Of the 30–60 P copies present in a D. melanogaster P strain genome, one third are complete P elements (BINGHAM et al. 1982 ; O'HARE and RUBIN 1983 ; O'HARE et al. 1992 ). These elements can produce the transposase required for their mobility (KARESS and RUBIN 1984 ; RIO et al. 1986 ) and are therefore autonomous. The other two thirds are defective in transposase synthesis (O'HARE et al. 1992 ); however these nonautonomous elements can be mobilized in trans by complete P elements (ENGELS 1984 , ENGELS 1989 ).

The repression ability of a P element depends on its structure and its insertion site (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; RASMUSSON et al. 1993 ). We have previously sought to identify regulatory P elements in the chromosomes of natural populations (RONSSERAY et al. 1989 ; BIEMONT et al. 1990 ) because in these populations selection has probably retained efficient regulatory copies, i.e., the right structure at the right site. We have genetically isolated, in a genomic context devoid of other P elements, a pair of autonomous P elements inserted at the telomere of an X chromosome (cytological site 1A on the polytene chromosomes) from a Russian natural population (RONSSERAY et al. 1991 ). In the germline, this line ["Lk-P(1A)"] can completely repress P-induced hybrid dysgenesis, P transposition and transcription from a P-lacZ transgene (RONSSERAY et al. 1991 , RONSSERAY et al. 1996 ; LEMAITRE et al. 1993 ). The repression ability of the Lk-P(1A) line is maternally transmitted. The P elements in this line are inserted in Telomeric Associated Sequences (TAS) (RONSSERAY et al. 1996 ) described by KARPEN and SPRADLING 1992 . These sequences have the properties of heterochromatin, including the ability to silence transgenes inserted within them. In addition, the repression ability of the P(1A) elements is sensitive to the dosage of HP1, a nonhistone heterochromatin protein (JAMES and ELGIN 1986 ; WUSTMANN et al. 1989 ), because heterozygosity for a null allele of the Su(var)205 locus, which encodes HP1, strongly impairs this ability (RONSSERAY et al. 1996 , RONSSERAY et al. 1997 ). HP1 has been shown to bind to centromeres and telomeres (JAMES and ELGIN 1986 ; JAMES et al. 1989 ). The repression ability of the Lk-P(1A) line probably requires the expression of the P(1A) elements because genetic analysis reveals a maternally transmitted component of repression termed the "P pre-cytotype" (RONSSERAY et al. 1993 ). The P(1A) elements are expressed because transposase activity can be detected genetically (RONSSERAY et al. 1996 ) and transcripts can be detected by RT-PCR (ROCHE et al. 1995 ).

However, the repression ability of the Lk-P(1A) line might not result exclusively from the expression of its P elements. A case of homology-dependent transgene silencing has been reported in tobacco (VAUCHERET 1993 ). In this study, a plant number "271" carried a telomeric silencer locus composed of multiple copies of two chimeric transgenes: an antisense nitrite reductase cDNA (RiN) under the control of the 35S promoter of cauliflower mosaic virus (CaMV 35Spro) and a neomycin-phosphotransferase gene under the control of the CaMV 19S promoter. This telomeric silencing locus is able to silence any euchromatic gene under the control of the 35S promoter (VAUCHERET 1993 , VAUCHERET 1994 ). The authors hypothesize that the silencing capacity of the telomeric 271 locus is due to the location of this locus. The telomeric position would allow a rapid scan of the entire genome and the silencer locus could recognize and inactivate a target at any genomic location.

Further, the paper by S. E. ROCHE and D. C. RIO (1998, this issue) provides evidence in favor of such a model in Drosophila. They found that a P-lacZ element located at the tip of the X chromosome can exert a trans-silencing effect: it represses the expression of a P-vasa-lacZ construct (described in ROCHE et al. 1995 ) located on an autosome. The silencing effect appears independent of the insertion site of P-vasa-lacZ. They call this effect "trans-silencing." We have observed a similar trans-silencing also using a P-lacZ at 1A but with a P-lacZ transgene as the euchromatic autosomal target instead of P-vasa-lacZ (S. RONSSERAY and L. MARIN, unpublished results). ROCHE and RIO 1998 propose that the trans-silencing occurs via a pairing between the P-lacZ located at 1A and the P-vasa-lacZ located on the autosome.

However, when we cross these P-lacZ(1A) females with P males (for example Harwich-2 males), the female offspring are almost completely sterile [>98% gonadal dysgenesis (GD); see Table 1], showing that P-lacZ(1A) lines are not equivalent to P(1A) lines carrying autonomous P elements at 1A, probably because they lack a P-encoded repressor. We have tested whether telomeric P-reporter insertions combined with other P elements can elicit significant P-repression capacities in crosses in which neither component alone can do so. We show in this article, that a P reporter at the X or 3R telomere can contribute to P repression when placed in hybrid females with P chromosomes that carry regulatory P elements. Telomeric P sequences, unable to encode a repressor, can therefore play a role in P-element repression.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
Canton^y is a typical M line (KIDWELL et al. 1977 ) containing no P elements and marked with a spontaneous allele of yellow. Sevelin and Oregon are two M lines. Muller5 is an M line with the Basc balancer chromosome, marked with Bar and w^a (LINDSLEY and ZIMM 1992 ). E24 is a line deriving from Lk-P(1A) that has only one of the two P elements at 1A (RONSSERAY et al. 1996 ). Exe24 is a line deriving from E24 that has lost the P(1A) element: it is therefore a neo-M line. Ch-P(1A) is a line with a single autonomous P element at 1A from a French natural population (RONSSERAY et al. 1996 ). Saltillo-P(1A) [abbreviated "Salt-P(1A)"] is a line with a defective P element at 1A deriving from a Mexican population (L. MARIN and S. RONSSERAY, unpublished results). Harwich-2 is a P line; the subline used here shows more than 80 P labels by in situ hybridization (11/12 larvae tested had no P label at 1A). It has an unidentified autosomal recessive marker (sepia-colored eye) that appeared in our Harwich stock. Tautavel 67 is a strong P strain collected in France (1967) that shows 40–50 P labels by in situ hybridization; no P element has been found at 1A (nine larvae tested; S. RONSSERAY and M. LEHMANN, unpublished results). Texas 007/Cy is a P line that has strong P repression ability linked to a wild derived second chromosome called "Texas 007" (PERIQUET and ANXOLABEHERE 1982 ); it shows 20–25 P labels including one at the 1A site (three larvae tested). The properties of these strains in regard to P-induced hybrid dysgenesis are given in Table 1. w^m4; Su(var)2-5⁰⁴/Cy Roi: this Su(var)205 allele encodes a truncated nonfunctional HP1 protein (EISSENBERG et al. 1992 ). It strongly impairs the repression ability of Lk-P(1A) (RONSSERAY et al. 1996 , RONSSERAY et al. 1997 ). A number of P-reporter insertions at various locations in the genome were used: the insertion sites are given in Table 1 Table 2 Table 3 Table 4 Table 5 Table 6. The structure of the transgene is indicated further in Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 by the two first letters of the name and is as follows: WG = P{ry[+t7.2] = 1ArB} = P-lacZ, ry, adh, from WALTER GEHRING; GR = P{w[+t11.7] ry[+t7.2] = wA} = P-w, ry, from GERRY RUBIN; GR' = P{w[+tAR] ry[+t7.2AR] = wA[R]} = P-w, ry, from GERRY RUBIN; JLC = P[lac-ry+]A = P-lacZ, ry, from JEAN-LOUIS COUDERC; LW = P{hsp26-pt-T hsp70-w+}, P-barley, w, from LORI WALLRATH; and DP = P{ry[+t7.2] = 23.1} = P-ry, from DANIEL PAULI.

Gonadal dysgenesis assays (%GD A*):
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 test cross, 3–10 pairs were mated en masse and immediately 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. Twenty-five 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, calculated as %GD = %S0 + %S1, will be referred to as the 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.

The ability of lines to induce dysgenic sterility was measured by the "A assay" (KIDWELL et al. 1977 ). This assay reflects the activity of autonomous P elements in the tested line. Males of the tested line were crossed with M females (Canton^y). For each test cross, 5–10 pairs were mated en masse and immediately placed at 29°. Parents were discarded after 3 days of egg laying. The percentage of GD sterility was measured as in the A* assay (see above).

Interactions between P-reporters and P genomes:
Maternal and zygotic contributions of the P-reporter: Five females of the line tested (P-reporter or control M line) were crossed at 20° to five males that carry regulatory P elements. Different males were used depending on the experiment [Lk-P(1A), Harwich-2, Texas 007/Cy and Tautavel 67]. Replicate sets of three to five G₁ females were then crossed with five Harwich-2 males at 29°. Parents were discarded after 3 days of egg laying and the percentage of GD sterility was estimated in their progeny (see the A* assay).

Zygotic contribution of the P-reporter: Five males of the line tested were crossed to five E24 females at 20°. Replicate sets of three to five G₁ females were crossed to five Harwich-2 males at 29°. Parents were discarded after 3 days of egg laying and the percentage of GD sterility was estimated in their progeny (see the A* assay).

G₁ males from the "maternal and zygotic component" of the previous assays were also tested for their ability to induce GD sterility when crossed with M females (A assay). Five to 10 G₁ males were crossed with 10 Canton^y females at 29°. Parents were discarded after 3 days of egg laying and GD sterility was measured in their daughters after dissection, as in the A* assay.

The sensitivity of the combination effect to Su(var)205 was also tested. The mating scheme is presented with the results in Figure 1.

View larger version (29K): In this window In a new window Download PPT slide   Figure 1. Sensitivity of the combination effect to Su(var)205 mutations. Su(var)205 is located on the second chromosome. The chromosome with the Su(var)205+ allele is marked with the Cy dominant mutation. The repression ability of the G1 was tested by crossing replicate sets of three to five G1 females with five P strain males (Harwich-2) at 29°. For each replicate test cross, 50 to 100 ovaries were examined in the progeny. m, s, n, see legend to Table 1; M5, Muller5; P-Z(1A), P-lacZ(1A) (WG-1103 insertion); 2054, Su(var)2-504.

Statistical analysis: The repression abilities of G₁ females from experiments described above were compared using the nonparametric Mann-Whitney U-test performed on A* assay replicates.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Combination effect of telomeric P-reporters with regulatory P elements:
Maternal and zygotic component: We investigated the properties of two lines carrying an insertion of a P-lacZ fusion transgene at the 1A site. These lines (WG-1103 and WG-1152) were chosen from among a collection of enhancer-traps established in WALTER GEHRING's laboratory (WILSON et al. 1989 ). Neither exhibited any lacZ staining in the ovaries even after an overnight reaction (S. RONSSERAY and L. MARIN, data not shown). Both WG-1103 and WG-1152 are flanked by TAS (ROCHE and RIO 1998 ).We crossed WG-1103 and WG-1152 females with Harwich-2 males at 29° and found 95–100% of GD sterility (Table 1), a level similar to that obtained when crossing M line females to Harwich-2 males under similar conditions. Therefore these P-lacZ(1A) insertions do not themselves repress dysgenic sterility. To test for an indirect effect of these P-lacZ(1A) insertions on P regulation, WG-1103 or WG-1152 females were crossed with Lk-P(1A) males at 20° and the repression ability of G₁ females was evaluated by the A* assay, i.e., by crossing them with Harwich-2 males at 29° and scoring GD sterility in the G₂ females. As a control, the same experiment was carried out with M females instead of P-lacZ(1A) females in the G₀ and also with females from two different lines [E24 and Ch-P(1A)] that have autonomous regulatory P elements at 1A (RONSSERAY et al. 1996 ).

Among the M lines used as a control, some have X-linked TAS, as tested by in situ hybridization, whereas others apparently lack such sequences (L. TOLAR, F. SHEEN and R. LEVIS, personal communication; L. MARIN and S. RONSSERAY, unpublished results). Starting the experiment with M females in the G₀, the G₁ females had no detectable repression ability (Table 2, first column). This result is expected since the Lk-P(1A) elements were paternally introduced in the G₀ and the repression ability of these elements is known to be maternally inherited (RONSSERAY et al. 1991 , RONSSERAY et al. 1996 ). Whether or not the M lines have X-linked TAS, there is a similar lack of repression ability in the G₁ females, indicating that the presence of the X-linked TAS does not affect the results of this assay. By contrast, using WG-1103 or WG-1152 females in the G₀ produced G₁ females with increased repression ability (Table 2, first column). Each comparison between the results of a given M line and those of WG-1103 or WG-1152 is significant (P < 0.01). "P(1A)" females [carrying autonomous P elements at 1A from the E24 or Ch-P(1A) lines] produced G₁ females with nearly complete repression ability. This resulted because these lines repress the occurrence of GD sterility by themselves (Table 1). However, the case of Salt-P(1A) is different. It has a naturally occurring defective P element at 1A and by itself does not repress GD sterility (Table 1). When Salt-P(1A) females are crossed to Lk-P(1A) males in the G₀, their G₁ daughters have significant but incomplete repression ability, similar to the G₁ daughters of by P-lacZ(1A) females crossed to Lk-P(1A) males.

These results show that: (1) a maternally inherited P-lacZ(1A) insertion alone is unable by itself to repress GD sterility; (2) paternally inherited regulatory P elements at 1A alone are also unable to repress GD sterility; (3) the combination of a maternally inherited P-lacZ insertion and paternally inherited regulatory P elements at 1A significantly represses GD sterility (there is therefore a "combination effect"); and (4) this regulatory effect can also occur when a naturally occurring defective P element at 1A is combined with paternally inherited regulatory P elements at 1A. It should be noted, however, that this combination effect is weaker than the effects obtained with maternally inherited complete P elements at 1A (as in the E24 or Ch-P(1A) lines; Table 2).

We also tested whether these P-lacZ(1A) elements can present a combination effect with regulatory P elements that are not located at 1A, but that are scattered throughout the genome. The Harwich-2 line, as recently tested by in situ hybridization, carries approximately 80 P elements per haploid genome. A P label at 1A was found in only 1 slide out of 12 analyzed, but at least two autosomal telomeric labels were observed on each slide (S. RONSSERAY and M. LEHMANN, unpublished results). In the G₀, we crossed females from the P-lacZ(1A), M, or P(1A) lines with Harwich-2 males [instead of Lk-P(1A) males as in the previous assay] at 20°. There is no GD sterility in the progeny at this temperature of development. Then we tested the repression ability of the G₁ females as above. When M females were used in the G₀ crosses, their G₁ daughters had a negligible or, at best, weak ability to repress GD sterility (Table 2, second column). By contrast, when WG-1103 or WG-1152 females were used in the G₀ crosses, strong repression ability was seen in their G₁ daughters. This effect was also observed when P(1A) females were used in the G₀ crosses even when the G₀ females come from the Salt-P(1A) line. Each comparison between results from a given M line and those from WG-1103, WG-1152, or a P(1A) line is significant (P < 0.01). These results show that the combination of a maternally inherited P-lacZ insertion at 1A and paternally inherited P elements at locations other than 1A (including euchromatic and autosomal telomeric locations) represses GD significantly.

Zygotic component: When Lk-P(1A) females are crossed to M males in the G₀, their G₁ daughters usually have strong repression ability because of the maternal inheritance of the Lk-P(1A) regulatory factors (RONSSERAY et al. 1991 ). E24 has only one of the two P elements present in Lk-P(1A) and its repression ability is accordingly weaker than that of Lk-P(1A) (Table 1; see also RONSSERAY et al. 1996 ). Crossing E24 females with M males produces G₁ females that have negligible repression ability (Table 3). Because these hybrid females from E24 mothers cannot repress GD sterility, we can investigate the effect of combining a paternally inherited P-lacZ(1A) element with a maternally transmitted P element from the E24 line. P-lacZ(1A) males were crossed with E24 females and the repression ability of the G₁ females was measured by the A* assay as in the previous experiments. Using either WG-1103 or WG-1152 males in the G₀ cross, we find that their G₁ daughters have significant repression ability (Table 3). Each comparison between the results from a given M line and those from WG-1103 or WG-1152 is significant (P < 0.01). As in the first set of experiments (Table 2), these repression properties are weaker than those obtained with autonomous P elements at 1A [E24 or Ch-P(1A)] instead of P-lacZ(1A) transgenes introduced in the G₀. These results show the contribution of the P-lacZ(1A) insertions to the combination effect can be at least partially transmitted through the male.

Effects of P-reporter constructs at other locations: The ability of P-reporter constructs inserted at other sites of the genome to induce a combination effect was investigated. None of these P-reporter lines had any significant P repression ability as tested by A* assay (data not shown). The capacity of these lines to influence the repression ability of P elements at 1A was investigated by crossing females carrying these P-reporter constructs to Lk-P(1A) males and testing their daughters by the A* assay. Among 15 euchromatic insertions tested, none resulted in a combination effect in the P-reporter/P(1A) hybrid females (Table 4). In addition, a "multi-lac" line that carries four P-lacZ insertions on the X chromosome also failed to produce a combination effect in the hybrid females. It must be pointed out that, among the euchromatic P-reporter insertions tested, some (BC69, BQ16 and ABOO) show very strong lacZ staining in the nurse cells and in the mature oocytes (J. L. COUDERC, personal communication), indicating that these P-reporter insertions are vigorously expressed in these tissues. By contrast, WG-1103 and WG-1152 are apparently not expressed in the nurse cells and in the oocyte (S. RONSSERAY and L. MARIN, unpublished results). Thus the P-reporter/P elements combination effect is not correlated with expression of the P-reporter insertion in the germline.

Among four insertions in pericentromeric heterochromatin that were tested, no case of combination effect was detected (Table 4). This includes the pericentromeric AS-CH(2)6 insertion which is flanked by TAS-related sequences (ZHANG and SPRADLING 1995 ). Among five insertions at an autosomal telomere that were tested, one (GR-833) produced a combination effect (Table 4). The GR-833 line carries a P-w, ry transgene located at 100F at the 3R telomere (HAZELRIGG et al. 1984 ). The combination effect with this insertion was also detected using Harwich-2 males in the G₀, i.e., GR-833 females x Harwich-2 males at 20°; less than 5% of GD sterility was found by the A* assay of the G₁ females from this cross (m = 3.5, s = 5.1, n = 8; compare with Table 2, second column). Because the GR-833 transgene (P-w, ry) does not include the lacZ gene, the combination effect does not depend on lacZ presence or expression nor does it require that the P-reporter be inserted on the X chromosome. The GR-833 insertion shows strongly variegated expression of its white transgene and it is flanked by TAS-related sequences (LEVIS et al. 1993 ). However, insertion in TAS-related sequences is not per se sufficient to produce a combination effect because the LW-39C5 insertion (WALLRATH and ELGIN 1995 ) and the WG-1155 insertion, neither of which produces a combination effect with Lk-P(1A), are also flanked by TAS-related sequences (L. WALLRATH, personal communication; ROCHE and RIO 1998 ).

The combination effect is seen in different P strain genetic backgrounds:
We tested for a combination effect between selected telomeric P-reporter insertions and the P elements from two independent P strains (Texas 007/Cy and Tautavel 67). In situ hybridization with a P probe showed that these two lines have numerous P labels scattered through the genome (MATERIALS AND METHODS). With Tautavel 67, there was no P label at 1A in the nine slides analyzed; however, 1–2 autosomal telomeric sites were labeled on each slide. With Texas007/Cy, a P label was seen at 1A and at the 3R telomere (100F) in the three slides analyzed. In the G₀, females from the telomeric P-reporter lines were crossed with males from the P strains (Texas 007/Cy or Tautavel 67) and the repression ability of their G₁ daughters (Cy⁺ phenotype) was measured by the A* assay. When M line females (Canton^y, Muller5, Oregon) were crossed in the G₀ with Texas 007/Cy or Tautavel 67 males, partial repression ability was observed in the G₁ daughters (Table 5). By contrast, when G₀ females with a telomeric P-reporter were crossed with either Texas 007/Cy or Tautavel 67 males, stronger repression ability was seen in the G₁ daughters, indicating a combination effect. The level of repression was close to that obtained with Lk-P(1A) G₀ females. These results therefore show that the combination effect does not depend on a particular P strain background.

Telomeric P-reporter insertions do not affect the ability of males to induce hybrid dysgenesis:
Because telomeric P-reporters influence the repression ability of natural P elements in the genome, we tested whether they also affect the ability of these elements to induce GD sterility. P males were crossed at 20° to females with telomeric P-reporter insertions. Their G₁ sons were then crossed at 29° to M females (Canton^y) and the percentage of GD sterility in the G₂ daughters was estimated (A assay). These G₁ males have paternally inherited P elements and a maternally inherited telomeric P-reporter. As controls, the same experiment was carried out with M females instead of telomeric P-reporter females in the G₀. Table 6 shows that the presence of the insertions WG-1103, WG-1152 or GR-833 does not significantly affect the ability of the P elements inherited from Harwich-2 or Texas 007/Cy to induce GD sterility. Thus there is no combination effect on the ability to induce GD sterility.

The combination effect is impaired by a Su(var)205 mutation:
The repression ability of the Lk-P(1A) line is sensitive to Su(var)205 mutations (RONSSERAY et al. 1996 , RONSSERAY et al. 1997 ). Su(var)205 encodes HP1, a nonhistone heterochromatin protein (JAMES and ELGIN 1986 ; WUSTMANN et al. 1989 ) that binds the centromeres and the telomeres (JAMES and ELGIN 1986 ; JAMES et al. 1989 ). The presence of a null allele of Su(var)205 or of a truncated HP1 protein strongly impairs, in a dominant manner, the ability of the Lk-P(1A) elements to repress GD sterility. Neither the other Modifiers of variegation tested, nor Y-chromosome dosage, affects Lk-P(1A) repression ability (RONSSERAY et al. 1996 ). The effect of a Su(var)205 mutation on the combination effect was tested according to the mating scheme presented in Figure 1. The Su(var)2-5⁰⁴/Cy Roi line used in this experiment is devoid of P repression ability. The presence of the Su(var)2-5⁰⁴ allele was previously shown to dramatically affect the repression ability of Lk-P(1A) (RONSSERAY et al. 1996 ). G₁ females were constructed that carried the WG-1103 insertion (maternally inherited) and P elements from Harwich-2 (paternally inherited); these females were either heterozygous Su(var)205⁺/Su(var)2-5⁰⁴ or homozygous Su(var)205⁺/Su(var)205⁺ (as a control). The repression ability of the two kinds of G₁ females was tested by the A* GD assay. G₁ females with two Su(var)205 wild-type alleles had significant repression ability whereas their sisters carrying the Su(var)2-5⁰⁴ allele did not. The difference between the two genotypes is highly significant (P < 0.01). Thus Su(var)205 strongly affects the ability of the combination of a telomeric P-reporter and natural P elements to repress GD sterility.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The repression ability of P(1A) elements involves a combination of effects:
The repression ability of a P element depends on both its structure and its genomic position (ANXOLABEHERE et al. 1987 ; BLACK et al. 1987 ; DANIELS et al. 1987 ; PRESTON and ENGELS 1989 ; ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; SIMMONS et al. 1990 ; RONSSERAY et al. 1991 ; HIGUET et al. 1992 ; GLOOR et al. 1993 ; MISRA et al. 1993 ; RASMUSSON et al. 1993 ; ANDREWS and GLOOR 1995 ; CORISH et al. 1996 ; SIMMONS et al. 1996 ). A strong position effect exists at 1A since two copies of autonomous P elements are sufficient to elicit the complete P cytotype whereas M lines transformed by the autonomous P element may reach around 15 copies without having a strong repression ability (ANXOLABEHERE et al. 1987 ; DANIELS et al. 1987 ). The repression ability of autonomous P elements at 1A probably depends on an encoded product since Lk-P(1A) females transmit a strong maternally-transmitted component (pre-P cytotype) that promotes nearly complete P repression in progeny that inherit other regulatory P elements from their father (RONSSERAY et al. 1993 ). The Lk-P(1A) elements are known to be expressed because each of the two elements, when separated, shows transposase activity (RONSSERAY et al. 1996 ). In addition, ROCHE et al. 1995 have used RT-PCR to detect transcripts from these elements, including an incompletely spliced RNA which could lead to the production of a truncated transposase protein (P-66kD) in the germline; this protein has previously been shown to be a repressor of P activity (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ). However, the expression of the Lk-P(1A) elements cannot alone explain their strong repression ability because the expression is undetectable as judged by western analysis with an antibody against P proteins (S. E. ROCHE, S. MISRA and D. C. RIO, personal communication). It is therefore likely that a peculiar property because of the telomeric position is also involved in repression by the Lk-P(1A) elements. The trans-silencing described in tobacco by VAUCHERET 1993 , VAUCHERET 1994 , MATZKE et al. 1994 and PARK et al. 1996 suggests that a chromatin-chromatin interaction may take place between a telomeric transgene insertion and a euchromatic insertion. The article from ROCHE and RIO 1998 (this issue) provides data suggesting such an interaction: they have observed a trans-silencing effect (TSE) by telomeric P-transgenes, unable to encode a P repressor, on other P-transgenes in the genome.

TSE and the regulatory combination effect appear to be closely linked:
The TSE described by ROCHE and RIO 1998 (this issue) and the combination effect described here, present several similarities. They are both induced by P-reporters but only at telomeric positions. There is a correlation between the TSE and the combination effect: for example, GR-833 shows both phenomena whereas WG-1155 shows neither. In fact, out of five insertions tested for both assays, there is no case of an insertion showing one but not the other phenomenon. Among lines that induce these effects, there is also a correlation for the strength of the effects: WG-1152 and GR-833 are stronger for both TSE and the combination effect than WG-1103. The TSE is suppressed by Enhancer of zeste (E(z)) mutants that also impair the repression ability of Lk-P(1A) (ROCHE and RIO 1998 , this issue). The combination effect is suppressed by a Su(var)205 mutation (Figure 1) that also impairs repression by the Lk-P(1A) elements (RONSSERAY et al. 1996 ). Both Su(var)205 and E(z) are known to affect the structure of the chromatin. It will be interesting to test the effect of E(z) mutations on the combination effect and of Su(var)205 mutations on TSE. However, differences exist between the two sensitivities, since Su(var)205 mutations have an effect on regulation by Lk-P(1A) when paternally inherited whereas E(z) mutations have an effect only when maternally inherited.

An attractive model is that the combination effect induced by telomeric P-reporters on the P elements of P strains [HARWICH, TAUTAVEL 67, Texas 007/Cy or Lk-P(1A)] is a consequence of a TSE on these P elements. Under this model, it can be hypothesized that a reduced expression of these P elements (because of the TSE) leads them to produce mainly repressor instead of transposase. Such a model, supported by genetic data, has been proposed by LEMAITRE et al. 1993 (see also COEN et al. 1994 ) and reinforced at the molecular level by ROCHE et al. 1995 . Nevertheless a striking result remains unexplained: why does this trans-silencing not change the ability of males to induce dysgenesis? One explanation is that the trans-effects and the induction of GD sterility occur at different stages. TSE and the combination effect described here are observed in the female adult germline in which the maternal contribution of the P repression is elicited. By contrast, the crucial period for the induction of GD sterility by males takes place in the progeny at an earlier stage (embryo and first instar larvae; ENGELS 1989 ).

The capacity of a telomeric P-insertion to repress GD sterility depends on its coding capacity:
The structure of the P insertion at 1A still appears to be essential in regard to its ability to elicit, in the absence of other P elements, strong repression of GD sterility. Lk-P(1A), with two autonomous P elements, completely represses GD sterility; E24, E60 and Ch-P(1A) each with one element partially repress it (RONSSERAY et al. 1996 ). By contrast, the lines WG-1103, WG-1152 and GR-833 show no repression of GD sterility. In addition, Salt-P(1A), which carries a naturally occurring defective P element at 1A (L. MARIN and S. RONSSERAY, unpublished results) that is apparently unable to encode the P repressors described so far, i.e., the P-66-kD truncated protein (ROBERTSON and ENGELS 1989 ; MISRA and RIO 1990 ; GLOOR et al. 1993 ) and the KP protein (BLACK et al. 1987 ; ANDREWS and GLOOR 1995 ; LEE et al. 1996 ), induces a combination effect but does not elicit significant repression of GD sterility by itself. Thus it behaves like a P-lacZ(1A) insertion. Furthermore, in the genetic experiments described in this article, autonomous P elements at 1A showed stronger levels of repression in the combination effect assays than telomeric P-reporter insertions or the Salt-P(1A) element. Clearly, P-LacZ insertions or defective P elements at 1A are not equivalent to autonomous P elements at this site. These differences are probably the consequence of the capacity of autonomous P elements at 1A to make a repressor.

The combination effect does not depend on telomeric P-transgene coding capacity:
The results of this article indicate that the ability of P-lacZ(1A) insertions to induce a combination effect is not correlated to expression of the transgene in the germline. In fact, the combination effect and TSE appear to be independent of the coding capacity of the telomeric P insertions and in particuliar do not depend on the presence of lacZ. Indeed the WG-1103 and WG-1152 insertions have a structure differing from that of GR-833. The GR-833 line carries a P-w, ry transgene and not lacZ. This shows that 587 bp of 5' P sequence and 233 bp of 3' P sequence are sufficient to induce the effect. It is possible that the P sequences flanking the transgenes are reponsible for the postulated interactions between telomeric and euchromatic P insertions. It would be interesting to test whether removing the P-sequences flanking one side of the transgene would abolish the combination effect and TSE.

The TAS adjacent sequences are perhaps necessary but not sufficient for the induction of the combination effect:
The combination effect (Table 4) and TSE can be induced by a P-reporter at an autosomal telomere (3R), showing that these properties are not restricted at the X chromosome telomere. However, the GR-833 insertion in 100F is also flanked by TAS-related sequences (LEVIS et al. 1993 ) and thus three out of three telomeric transgenes, which can induce the combination effect and TSE, are in TAS or TAS-related sequences. In addition three out of three naturally occurring P elements at 1A, which have the capacity to repress dysgenic sterility, are inserted in TAS. This suggests that TAS are necessary for the combination effect and consequently for P repression to occur. However, the WG-1155 insertion (which has the same P-reporter structure as WG-1103 and WG-1152) and the LW-39C5 insertions are also inserted in TAS-related sequences (ROCHE and RIO 1998 ; L. WALLRATH, personal communication) but they do not induce the combination effect or TSE. This shows that TAS-related adjacent sequences are not per se sufficient to induce these effects. One explanation may be that the size of the TAS-cluster differs between the insertions tested and is not sufficient in lines that fail to induce the combination effect and TSE (WG-1155, LW-39C-5). Another explanation is that the position of the P-transgene inside the TAS-related-repeats cluster differs in lines able or unable to induce the combination effect and TSE. Finally, it is possible that TAS have no functional role in the repression ability of telomeric P-insertions and that the apparent correlation is a consequence of the fact that TAS are hot spots for P insertions (KARPEN and SPRADLING 1992 ).

The combination effect: a chromatin structure effect or a nuclear location effect:
It appears that at least part of the ability of the P elements at 1A to repress hybrid dysgenesis and to cause TSE involves some coding-independent capacity to interact with other P elements. To explain TSE, ROCHE and RIO 1998 , propose that a pairing occurs between P-reporter insertions at 1A and other P-reporter insertions in the genome. To explain the combination effect, we propose a similar interaction between telomeric P reporters and natural P elements scattered through the genome. The combination effect would be a trans-stimulation by the telomeric insertions of the P repression elicited by the P elements of P strains. The latter P elements could be copies at 1A (Lk-P(1A)). They could also be euchromatic copies or copies located at autosomal telomeres since Harwich-2 and Tautavel 67 have both types of P elements (but no copies at 1A). Texas 007/Cy has P copies at 1A, at 100F and in euchromatin: each of them could play a role in the combination effect. The pairing would be facilitated by a scanning of the genome by the telomeric insertions as has been proposed to occur in plants. This pairing could be promoter-dependent as in tobacco (VAUCHERET 1993 ). Because of this pairing, the other P elements or reporters in the genome would be repressed as suggested by TSE. This repression could be the consequence of a trans-heterochromatinization of the target P elements, since P-insertions at the telomeres are inserted in heterochromatic TAS sequences. An alternative possibility is that the telomeres are located in a special compartment of the nucleus. The relocalization of the other P elements or reporters in the genome would lead to their repression since the nuclear compartment may be important in regard to gene expression (WAKIMOTO and HEARN 1990 ). It will be interesting to analyze the P insertions at 1A and the other P insertions in the genome for the chromatin structure by accessibility to restriction enzyme digestion (WALLRATH and ELGIN 1995 ) and for their location in the nucleus by FISH. Similarities between animal and plant transposons have recently been revealed by the discovery of a "cosuppression-like" phenomenon in Drosophila (PAL-BADHRA et al. 1997); the correlated combination and trans-silencing effects by P insertions at Drosophila telomeres mimic (at least partially) the telomeric transsilencing in tobacco (VAUCHERET 1993 , VAUCHERET 1994 ; MATZKE et al. 1994 ; PARK et al. 1996 ) and suggest that transposable elements have essential similarities in animals and plants.

	ACKNOWLEDGMENTS

We thank J. L. COUDERC, A. SPRADLING, D. DORER, S. HENIKOFF, L. WALLRATH, F. SHEEN, L. TOLAR, R. LEVIS, S. ROCHE, S. MISRA and D. RIO for providing lines and/or for sharing unpublished information. We thank S. ROCHE for helpful comments. We thank MATHEW COBB for assistance in the preparation of the manuscript. We thank the reviewers for their helpful comments and suggestions. We thank the Bloomington and Umea stock centers for providing stocks including numerous P-reporter lines and Flybase for helpful information. This work was supported by the Centre National de la Recherche Scientifique and by the Universités Paris 6-Pierre et Marie Curie and Paris 7-Denis Diderot (Unité Mixte de Recherche 7592, Institut Jacques Monod, Dynamique du Génome et Evolution).

Manuscript received October 23, 1997; Accepted for publication April 30, 1998.

	LITERATURE CITED

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