Construction of the Flipper 2 element:

The construction of Flipper 2 (see Figure 1A) was a multi-step cloning procedure. Details can be obtained upon request. In brief, the backbone of Flipper 2 consists of the intronless yellow gene (referred to as Dint in Geyer and Corces 1987) cloned into the P-element vector Carnegie 4 (Rubin and Spradling 1983). In this plasmid (from here on referred to as C4yellow), the wing and body-color enhancers are located 5′ of the yellow cDNA. It was kindly provided by Pam Geyer. The mini-white gene (Pirrotta 1988) was introduced into the SalI- and XbaI-restricted C4yellow as an XhoI–XbaI fragment. The resulting plasmid is called pC4YM. This P-element vector contains unique XhoI and NotI sites on the 5′ side of the mini-white gene. These two sites were used to introduce an XhoI–NotI fragment consisting of two parts, one of which is the 661-bp NdeI–PstI bxd element (Sigrist and Pirrotta 1997) flanked by FRT sites (Golic and Lindquist 1989). The FRT-bxd-FRT cassette was excised from plasmid pBSscriptII+FPREF, which was kindly provided by Christian Sigrist. The other part is the 2.9-kb EcoRI Mcp element (Müller et al. 1999) flanked by LoxP sites (Siegal and Hartl 1996). The orientation of Mcp is such that the end normally adjacent to iab-4 is closer to FRT-bxd-FRT.

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Figure 1.—

Insertion of an Mcp-containing P element in the apterous regulatory region. (A) A schematic of the Flipper 2 element (top). ap^MM-Mcp-bxd is an insertion of the Flipper 2 element 403 bp upstream of the ap transcriptional start site (D. melanogaster Genome Release 5.1 coordinates 1614738). (B) Wing phenotypes of the ap^MM-Mcp, ap^MM, and ap^MM-bxd inserts. ap^MM-Mcp homozygotes have wing defects (N ≥ 684). (C) The following method was used for scoring the severity and penetrance of the ap phenotype: class 1, wild-type wings; class 2, mild-to-severe notching; class 3, approximately normal amount of wing tissue present, but wing blistered or crumpled; class 4, strap-like wings; and class 5, very little or no wing tissue. (D) A representative ap^MM-Mcp homozygous (class 3) wing. (E) When the Mcp element is deleted, the wings of ap^MM flies are completely wild type. (F) Expression of white in ap^MM-Mcp wing discs. Because ap expression is disrupted by the Mcp insert, the wing disc is reduced in size and malformed; however, white expression is evident. (G) In ap^MM-Mcp/+ wing discs, white is clearly expressed in the ap pattern.

P-element-mediated transformation:

Flipper 2-containing transgenic lines were generated according to standard procedures (Spradling and Rubin 1982). DNA was co-injected along with the P-turbo helper plasmid into Df(1)w^67c23, y⁻ embryos. Transformants were detected by rescue of the white⁻ eye-color phenotype and/or the rescue of the yellow⁻ body-color and wing phenotypes. A total of 30 independent Flipper 2 lines, which will be described in more detail elsewhere (M. Müller, I. Hogga and V. Pirrotta, unpublished results), were established. One of these lines (isolation no. 81.38.2) was found to be inserted in the apterous gene and from here on will be referred to as ap^MM-Mcp-bxd. The mini-white reporter is dominantly suppressed in ap^MM-Mcp-bxd flies and its derivatives, but the insert could be identified thanks to strong yellow⁺ expression in the wings and variegated expression in the adult abdominal cuticle. In the abdominal cuticle, yellow⁺ expression is dosage dependent. There is no sign of pairing-dependent silencing.

Deletion of Mcp and bxd from the Flipper 2 element:

yw;ap^MM-Mcp-bxd/SM6a females were crossed with yw;TM6B P[w⁺, cre]/MKRS, hsFLP males (Siegal and Hartl 1996; stock obtained from Francois Karch). The progeny of this cross were heat-shocked twice for 1 hr during late embryogenesis and the first instar larval stage. Among the emerging adults, yw; ap^MM-Mcp-bxd/+;TM6B P[w⁺, cre]/+ and yw; ap^MM-Mcp-bxd/+; MKRS, hsFLP/+ males were collected and crossed with yw;l(2)/SM6a virgins. The progeny of these two crosses were screened for loss of Mcp or loss of bxd, respectively. On the basis of experience with other Flipper 2 transgenes, a change in the expression of the yellow and/or the mini-white reporter gene was expected. However, compared to ap^MM-Mcp-bxd/+ control flies, apart from a moderate increase in yellow expression on the abdomen, no striking differences were apparent. Therefore, a number of single putative yw;ap^MM-Mcp/SM6a and yw;ap^MM-bxd/SM6a males were selected and independent stocks were established. The presence of a deletion chromosome was confirmed with the following PCR reactions: ap^MM-bxd, primer 1 (mini-white 5′, AAGGCGGACATTGACG) and primer 2 (5328, TGGAGTACGAAATGCG). On an agarose gel, the loss of Mcp is accompanied by the change of a 4.5-kb band to a 1.3-kb band: ap^MM-Mcp, primer 1 (miniwhite 5′, see above) and primer 2 (Mcp22/7, CTTCCCTTTCCGAGCG). On an agarose gel, the loss of bxd is accompanied by the change of a 1.26-kb band to a 0.42-kb band.

ap^MM was made by deleting bxd from ap^MM-bxd. Briefly, 0- to 24-hr embryos from P{hsFLP}12, y¹ w* (from BL#1929); ap^MM-bxd/CyO flies were collected in bottles, allowed to age for 24 hr, and then heat-shocked for 1.5 hr/day in a 38° water bath until the majority of larvae had formed pupae. Heat-shocked P{hsFLP}12, y¹ w*; ap^MM-bxd/CyO females were collected and crossed to yw; bTf/CyO males and multiple stocks were established and screened for the loss of bxd by PCR. The following primers were used to confirm the deletion of bxd in ap^MM: primer 1 (8-2, TGTTCAGATGCTCGGCAGATGG) and primer 2 (PEP5′in, GTGACTGTGCGTTAGGTCCTGTT).

Determining the insertion site of ap^MM-Mcp-bxd by inverse PCR:

Inverse PCR was performed as previously described Bellen et al. (2004), with several minor modifications. Briefly, DNA was isolated from ap^MM-Mcp-bxd flies. A total of 50 flies were frozen in liquid nitrogen, homogenized in lysis buffer (0.1 m Tris–HCl, pH 8.0, 0.4 m NaCl, 25 mm EDTA, 1% SDS), mixed with an equal volume of Tris-buffered phenol, and centrifuged to remove debris. The supernatant was then phenol–chloroform extracted three times, washed once with chloroform, ethanol precipitated, and resuspended in 100 μl dH₂O. After treatment with 10 μg of RNaseA for 1 hr at 37°, 10 μl of ap^MM-Mcp-bxd DNA was digested with HinP1I (New England Biolabs, Beverly, MA). The HinP1I enzyme was heat inactivated by incubation at 65° for 30 min, and the sample was diluted 40-fold and ligated with T4 DNA ligase (New England Biolabs). The ligation reaction was carried out overnight at 4° to favor intramolecular ligation. DNA was isolated from the ligation reaction by ethanol precipitation and amplified by nested PCR using the following primers: 5′-end, PCR 1 (plac1, CACCCAAGGCTCTGCTCCCACAAT, and pwht1, GTAACGCTAATCACTCCGAACAGGTCACA); 5′-end, PCR 2 (sp1, ACACAACCTTTCCTCTCAACAA, and pwht1, see above); 3′-end, PCR 1 (pry1, CCTTAGCATGTCCGTGGGGTTTGAAT, and pry4, CAATCATATCGCTGTCTCACTCA); and 3′-end, PCR 2 (pry2, CTTGCCGACGGGACCACCTTATGTTATT, and pry1, see above).

The PCR products were excised from a gel and isolated using the QIAquick gel extraction kit (QIAGEN, Valencia, CA). The PCR products were then sequenced using the sp1 (5′) and pry2 (3′) primers. The insertion site of ap^MM-Mcp-bxd was determined to be 403 bp upstream of the apterous transcriptional start site (D. melanogaster Genome Release 5.1 coordinates 1614738). The insertion site and orientation were confirmed by PCR and sequencing between sp1 and apPromR and between plac1 and apPromR:

apPromR, TGGTCTGCAGCTGATCTA.

Scoring the apterous wing phenotypes:

In general, crosses were set up between five to six virgin females and three to four males. Two to three vials were set up in duplicate (for a total of four to six vials). These replicates were compared to calculate a standard deviation. Flies were allowed to lay eggs for 4 days and then the crosses were brooded into new vials. Wing phenotypes were scored each day, until all of the flies in each vial had eclosed. Individual wings were given a score from 1 to 5 on the basis of the severity of the wing defect (for representative wings, see Figure 1C). Wings that were wild type or that had only very minor bristle or wing-vein defects were scored as class 1. Wings with mild-to-severe notching were scored as class 2. Wings that were of approximately normal size, but were blistered or crumpled, were scored as class 3. Wings that were significantly reduced in size or were strap-like in appearance were scored as class 4. Finally, when little or no wing tissue was present, this was scored as class 5. All graphs depict the mean percentage of wings in each of the five classes. Error bars in each graph represent one standard deviation from the mean. Wing specimens shown in the various figures were dissected in 95% ethanol and mounted in Hoyer's medium. Pictures were taken using a Nikon DXM200F digital camera on a Nikon Microphot-SA light microscope.

In situ hybridizations:

In situ hybridizations were done as previously described (Tautz and Pfeifle 1989). Briefly, probes for white or yellow were prepared by in vitro transcription in the presence of digoxigenin (DIG)-labeled dNTPs (Roche). The probe for white was made using T7 polymerase from a white-containing plasmid obtained from Jumin Zhou. A portion of the yellow coding region was amplified by PCR using the following primers: yellow for (GGATTCCGGCCACTCTGACCTAT) and yellow rev (CTGGTCTGAGGTTTCTGTGGCAA).

The yellow PCR product was cloned into the pCRII-TOPO vector (Invitrogen, San Diego), and the yellow probe was made using SP6 polymerase. ap^MM-Mcp was balanced over CyO, P{w[+mC]=ActGFP}JMR1 (BL#4533) to select homozygous ap^MM-Mcp larvae. Homozygous ap^MM-Mcp or ap^MM larvae were selected and the imaginal discs and central nervous system (CNS) were dissected in PBS. Tissues were fixed in 4% formaldehyde in PBS for 20 min at room temperature, while rocking. The tissues were then washed thoroughly with phosphate-buffered saline + 0.1% Triton X-100 (PBST) and allowed to prehybridize in hybridization buffer (50% formamide, 5× SSC, 50 μg/ml heparin, 0.1% Tween 20, 100 μg/ml sonicated salmon sperm DNA) for 2 hr at 55°. The DIG-labeled probes were then diluted 1:100, heated to 80°, added to the tissue, and incubated at 55° overnight to hybridize. The probe was then removed and the sample was washed extensively with hybridization buffer, followed by PBST. The sample was then probed with 1:2000 HRP-conjugated anti-DIG antibody (Roche) for 1.5 hr. Upon removal of the antibody, the sample was washed extensively with PBST and then washed twice with developing solution (0.1 m NaCl, 0.1 m Tris–HCl, pH 9.0, 0.05 m MgCl₂, 0.1% Tween 20). The tissues in developing solution were transferred to a glass dish and 20 μl of solution [18.75 mg/ml Nitro blue tetrazolium chloride, 9.4 mg/ml 5-bromo-4-chloro-3′-indolyl phosphate, toluidine salt, in 67% dimethyl sulfoxide (w/v) (Roche)] was added. In situs were developed for between 30 and 60 min. The reaction was stopped by washing twice with PBST. Imaginal discs and brains were then mounted on slides in 70% glycerol and pictures were taken using a Nikon DXM200F digital camera on a Nikon Microphot-SA light microscope.

Reverting the ap^f00451 insertion by mobilizing the piggyBac element:

ap^f00451 virgins were crossed with w¹¹¹⁸; CyO, P{Tub-PBac\T}2/wg^Sp-1 (BL#8285) males. w; ap^f00451/CyO, P{Tub-PBac\T}2 males or females were selected from this cross and mated with w; Sp Pin/CyO virgins or males, respectively. A total of 19 independent crosses were set up. Among the progeny of these 19 crosses, ap^f00451revertant males with white eyes due to the loss of the PBac{WH} transposon were isolated and individually crossed with w; Sp Pin/CyO virgins. In this way, nine independent ap^f00451 revertant stocks were established. All nine stocks were homozygous viable and had normal wings.

Transvection at the apterous locus:

The fact that the ap phenotype of ap^MM-Mcp flies is due to Mcp enhancer blocking is further supported by the fact that interallelic complementation characteristic of transvection is observed when ap^MM-Mcp is crossed to other ap alleles. When ap^MM-Mcp is crossed to Df(2R)nap1, a deficiency that deletes the ap gene, the wing defects due to the Mcp insertion are unchanged (Figure 2, A and B). In contrast, when ap^MM-Mcp is crossed to ap^UGO35, a null mutation that deletes the ap transcriptional start site as well as the first exon (Cohen et al. 1992), the wing defects are strongly suppressed (Figure 2, A and C). The simplest explanation of this interallelic complementation is that the wing enhancer on the ap^UGO35 chromosome is able to act in trans on the ap^MM-Mcp chromosome, a phenomenon known as transvection (Figure 2C). It is also possible that the enhancer in cis to the Mcp boundary is able to bypass the boundary due to structural disruption of the ap locus when ap^MM-Mcp is crossed to ap^UGO35 (Figure 2C; Morris et al. 1998). And, while the wing defects of ap^MM-Mcp/ap^UGO35 are significantly less severe than those of ap^MM-Mcp homozygotes, the wings are not completely wild type. This would suggest that, in the presence of the Mcp boundary, the activation of ap in trans by the wing enhancer is less efficient than cis activation.

A piggyBac insertion containing the su(Hw) boundary element inserted in the apterous regulatory region:

A second boundary-element-containing insertion in the ap regulatory region was obtained from the Harvard–Exelixis stock collection (Parks et al. 2004; Thibault et al. 2004). This piggyBac WH element, ap^f00451, contains the su(Hw) boundary element and is inserted ~10.1 kb upstream of the ap transcriptional start site. ap^f00451 has a weak, but highly penetrant, ap phenotype (Figure 3, A and C). The wing defect of ap^f00451 is attributable to the presence of the piggyBac insertion, as nine of nine revertants obtained by mobilizing the piggyBac transposon are homozygous viable and have wild-type wings (data not shown). The insertion site of ap^f00451 is near the middle of an ~6-kb fragment that is capable of driving reporter gene expression in the ap pattern in the wing disc and CNS (Lundgren et al. 1995). The fact that ap^f00451 has a weak ap phenotype suggests that this insert is able to partially block the wing enhancer (perhaps blocking elements of the enhancer that are distal to the insertion site, but not affecting the gene proximal portions of the wing enhancer).

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Figure 3.—

A su(Hw)-insulator-containing P element also blocks the apterous wing enhancer and supports transvection. (A) As with ap^MM-Mcp, the wing defects of homozygous ap^f00451 flies and ap^f00451/Df(2R)nap1 flies are similar, but are strongly suppressed when ap^f00451 is crossed to ap^UGO35. The ap phenotype of ap^f00451 is also strongly suppressed by the su(Hw)^v/su(Hw)^f and mod(mdg4)^u1 mutants, which interfere with su(Hw) element enhancer-blocking activity (N ≥ 142). (B) ap^MM-Mcp is not affected by either su(Hw)^v/su(Hw)^f or mod(mdg4)^u1 mutants. This suggests that su(Hw) and mod(mdg4) are not involved in Mcp boundary activity; neither do they affect the normal regulation of the ap gene, so the effects on ap^f00451 seen in A, E, and F are specific to the su(Hw) boundary (N ≥ 72). (C) A representative ap^f00451 (class 3) wing. (D) A representative ap^f00451/ap^UGO35 (class 1) wing. (E) A representative ap^f00451; su(Hw)^v/su(Hw)^f (class 1) wing. (F) A representative ap^f00451; mod(mdg4)^u1 (class 1) wing. (G) A schematic depicting the chromosomes of ap^f00451/ap^UGO35. In this genotype, the trans and/or the cis wing enhancers are able to bypass the su(Hw) boundary. The ap gene is also likely activated by the unblocked, ap proximal portion of the wing enhancer (dashed arrow).

This suggestion is supported by analysis of the effects of mutations in two of the trans-acting factors that are required for enhancer blocking by the su(Hw) element. Both the Su(Hw) and Modifier of mdg4 [Mod(mdg4)] proteins are necessary for su(Hw) element enhancer blocking. Su(Hw) is a DNA-binding protein containing 12 zinc-finger domains, which binds to the YRTTGCATACCY repeats present in the su(Hw) element from the gypsy reterotransposon (Parkhurst et al. 1988; Geyer and Corces 1992; Parnell et al. 2006; Ramos et al. 2006). Mod(mdg4) is a BTB/POZ domain-containing protein that can interact with Su(Hw), other components of the su(Hw) insulator, and itself to form insulator bodies (Parkhurst et al. 1988; Geyer and Corces 1992; Gerasimova and Corces 1998; Gerasimova et al. 2000; Ghosh et al. 2001). To test whether the ap wing phenotypes observed in ap^f00451 flies are due to the presence of the su(Hw) boundary element, ap^f00451 flies were crossed to mutants in su(Hw) and mod(mdg4). When ap^f00451 was crossed to the hypomorphic combination su(Hw)^v/su(Hw)^f, the wing defects were nearly completely suppressed (Figure 3, A and E). Similarly, when ap^f00451 was crossed into a homozygous mod(mdg4)^u1 mutant background, the wing defects were also strongly suppressed (Figure 3, A and F). The wings of ap^f00451; mod(mdg4)^u1 flies are nearly wild type, with the exception of a disrupted L2 wing vein; however, this is likely due to the mod(mdg4) mutation rather than ap^f00451, as the wing-vein defect is also present in +/+; mod(mdg4)^u1 flies. The fact that both su(Hw)^v/su(Hw)^f and mod(mdg4)^u1 strongly suppress the wing defect of ap^f00451 suggests that, like ap^MM-Mcp, the phenotype of ap^f00451 is due to disruption of the ability of the wing enhancer to activate ap by the su(Hw) boundary element.

We also tested whether su(Hw) or mod(mdg4) mutations have any effect on the boundary activity of the Mcp element in ap^MM-Mcp. Neither su(Hw)^v/su(Hw)^f nor mod(mdg4)^u1 had an effect on the wing defects observed with ap^MM-Mcp, indicating that su(Hw) and mod(mdg4) do not affect the Mcp boundary, nor do they affect the regulation of ap in the absence of the ap^f00451 insert (Figure 3B). In addition, mod(mdg4)^u1 was crossed to the Beadex¹ (Bx¹) mutation. The Bx gene is a direct transcriptional target of ap, and the Bx¹ mutation has been used to screen for other genes involved in the regulation of ap (Milan et al. 2004). mod(mdg4)^u1 had no effect on the wing defects observed with Bx¹, indicating that mod(mdg4) is not normally involved in the ap pathway (data not shown).

As in the Mcp-containing insert, ap^MM-Mcp, interallelic complementation characteristic of transvection was observed for ap^f00451. The phenotype of ap^f00451/Df(2R)nap1 is as severe as that of ap^f00451 homozygotes (Figure 3A). As with the Mcp insert, the wing defect of ap^f00451 was strongly suppressed when ap^f00451 was crossed to the promoter deletion, ap^UGO35, suggesting that this allele is also able to support transvection (Figure 3, A, D, and G).

Deletion of the apterous wing enhancer—testing the transvection hypothesis:

To provide further evidence that transvection occurs at the ap locus, the region containing the ap wing enhancer was deleted by FLP-mediated recombination between FRT sites in ap^MM-Mcp and the insert PBac{RB}e01573 (Golic and Golic 1996a; Parks et al. 2004; Thibault et al. 2004). The resulting deletion, ap^DG-Mcp, deletes an ~26.8-kb region spanning the ap wing enhancer. ap^DG-Mcp is homozygous viable, indicating that it does not disrupt the function of the neighboring gene, l(2)09851, which is ~500 bp from the deletion breakpoint. As expected for an ap wing-enhancer deletion, ap^DG-Mcp homozygotes completely lack wings (Figure 4, A and C). Likewise, ap^DG-Mcp/Df(2R)nap1 flies fail to form wings (Figure 4A). However, robust interallelic complementation is seen between ap^DG-Mcp and ap^UGO35 (Figure 4, A and E). While neither ap^DG-Mcp nor ap^UGO35 homozygotes have any observable wing tissue, the majority of ap^DG-Mcp/ap^UGO35 flies have either class 3 (crumpled or blistered) or class 4 (strap) wings. To test whether the Mcp element in ap^DG-Mcp attenuates enhancer action in trans, we generated an ap wing-enhancer deletion derivative that lacks the Mcp element, ap^DG, using Cre recombinase. The transvection effect is much more striking when the Mcp element is excised from the enhancer deletion. ap^DG/ap^UGO35 flies have almost completely wild-type wings (Figure 4, B and F). The fact that transvection is stronger in ap^DG/ap^UGO35 flies compared with ap^DG-Mcp/ap^UGO35 flies indicates that Mcp is able to block the enhancer on the ap^UGO35 chromosome in trans. It is interesting to note that enhancer action in trans in the ap^DG/ap^UGO35 combination is sufficient for nearly wild-type levels of expression (Figure 4G).

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Figure 4.—

Deletion of the apterous wing enhancer. (A) Both the Mcp-containing enhancer deletion ap^DG-Mcp and ap^UGO35 are completely defective in forming wings either as homozygotes or over Df(2R)nap1. However, the wing defects of these mutants are significantly suppressed in ap^DG-Mcp/ap^UGO35 trans-heterozygotes (N ≥ 196). (B) As with ap^DG-Mcp, both homozygous ap^DG and ap^DG/Df(2R)nap1 flies fail to form any wing material. When the enhancer deletion ap^DG is crossed to ap^UGO35, >90% of the wings are completely wild type (N ≥ 158). (C) A homozygous ap^DG-Mcp fly. (D) A homozygous ap^DG fly. (E) A representative ap^DG-Mcp/ap^UGO35 (class 3) wing. (F) A representative ap^DG/ap^UGO35 (class 1) wing. (G) A model for the transvection observed in ap^DG/ap^UGO35 flies.

The role of the promoter in ap transvection:

We also tested whether transvection was observed for several additional ap mutations that, unlike ap^UGO35, are likely to have an intact ap promoter. Two spontaneous ap mutants, ap⁴ and ap^56f, the P-element insertion ap^rk568, and the piggyBac WH insertion ap^f08090 were tested for transvection in combination with the Mcp boundary insertion (ap^MM-Mcp), the enhancer deletion (ap^DG), and the enhancer deletion linked to the Mcp boundary (ap^DG-Mcp). While no molecular information is available for ap⁴ and ap^56f, it is likely that these mutants disrupt the ap-coding region, and not the regulatory elements, as they fail to complement ap^UGO35 (data not shown). ap^f08090 is an insertion in the second large intron of ap, just upstream of the ap-RB transcriptional start site (D. melanogaster Genome Release 5.1 coordinates 1597428). On the basis of complementation data and the fact that ap^f08090 is not suppressed by mutations in su(Hw) or mod(mdg4) (data not shown), the ap mutant phenotype observed with this allele is likely to be due to a disruption of the ap open reading frame (ORF), rather than enhancer blocking by the su(Hw) insulator present in the WH piggyBac transposon.

ap⁴, ap^56f, ap^rk568, and ap^f08090 all suppress the ap^MM-Mcp wing phenotype (Figure 5A). However, the suppression observed with these other four ap alleles is weaker than that seen when ap^MM-Mcp is crossed to ap^UGO35, suggesting that the ap wing enhancer can be tethered by an intact promoter in cis. On the other hand, since some transvection is still observed in these four mutants that likely do not disrupt the ap promoter, the cis tethering of enhancers at ap must be weaker than that observed at the endogenous y locus, where an intact promoter in cis largely suppresses transvection (Morris et al. 1999b; Lee and Wu 2006). ap^rk568 is an insertion of a P element 23 bp 5′ of the annotated ap transcription start site, suggesting that this insert may compromise, but not completely abolish, promoter function. Consistent with this observation, ap^rk568 supports transvection at a level intermediate to ap^UGO35 and the other ap alleles tested. Like ap⁴ and ap^56f, ap^rk568 fails to complement ap^UGO35 (data not shown).

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Figure 5.—

Transvection observed with other ap alleles. Several other ap alleles were tested for transvection effects: (A) As with ap^UGO35, ap^56f, ap^rk568, ap^f08090, and ap⁴, all exhibited transvection when crossed to the boundary insertion ap^MM-Mcp. These alleles fail to complement one another and Df(2R)nap1 or Df(2R)nap2 (data not shown). This suggests that, as with ap^UGO35, all these additional alleles affect the ap-coding region/promoter, and not the ap regulatory elements. Also, the transvection effect with ap^UGO35 is the strongest of all the alleles. This is likely because ap^UGO35 deletes the ap promoter, which releases the wing enhancer to act only in trans (N ≥ 406). (B) ap^56f, ap^rk568, ap^f08090, and ap⁴ also exhibit transvection when crossed to the Mcp-containing enhancer deletion ap^DG-Mcp. The transvection seen with these four alleles is weaker than that observed in ap^UGO35/ap^DG-Mcp flies (N ≥ 268). (C) ap^56f, ap^rk568, ap^f08090, and ap⁴ also exhibit transvection when crossed to the enhancer deletion ap^DG. The transvection seen with these four alleles is weaker than that observed in ap^UGO35/ap^DG flies (N ≥ 312).

We also tested combinations between ap^DG and the putative ap-coding region mutations that are expected to retain the promoter. As shown in Figure 5C, transvection is also observed when ap^DG is combined with these putative point mutations; however, the wing phenotype is not as completely suppressed as it is in the ap^DG/ap^UGO35 combination. Although the suppression seen when ap^DG is combined with these putative ORF mutations is not as strong as when it is combined with the promoter deletion ap^UGO35, the transvection effects with these alleles are considerably stronger than those observed when these alleles are combined with the enhancer deletion that retains the Mcp element, ap^DG-Mcp (Figure 5B). This again indicates that the Mcp element can partially interfere with trans-regulatory interactions.

Effect of zeste mutants on transvection at apterous:

Previous studies have implicated the Zeste protein in transvection at some, but not all loci. Transvection effects at white, yellow, Ubx, dpp, and eya are all sensitive to zeste (z) mutants (Lewis 1954; Gelbart and Wu 1982; Geyer et al. 1990; Leiserson et al. 1994; Duncan 2002). However, other instances of transvection that are insensitive to mutations in z, such as those observed at Scr, Abd-B, and vg (Hopmann et al. 1995; Southworth and Kennison 2002; Coulthard et al. 2005), have been identified. These observations, coupled with the fact that z null mutants are viable and do not have notably disrupted chromosome pairing (Goldberg et al. 1989; Pirrotta 1999), suggest that parallel or redundant mechanisms must exist for maintaining somatic chromosome pairing.

Two alleles of zeste, z¹ and z^a, were tested to see if they influenced transvection at the ap locus. The z¹ allele is a gain-of-function mutation that leads to hyperaggregation of the Zeste protein and thus generally increases the strength of a transvection effect (Pirrotta et al. 1987; Chen et al. 1992; Chen and Pirrotta 1993a,b). z^a is a hypomorphic mutation, which generally disrupts transvection (Goldberg et al. 1989). The z¹ mutant had little or no effect on transvection in ap^UGO35/ap^DG flies, while the z^a mutation caused only a slight disruption of transvection in this genotype (Figure 6A). However, stronger effects were seen when the zeste mutants were crossed to a pair of ap alleles in which transvection is less robust. In ap^56f/ap^DG flies, suppression of the wing phenotype was observed in the hyperaggregating z¹ mutant background, while little or no effect was seen with z^a (Figure 6B). This finding is similar to what was previously observed for transvection at the dpp locus, where effects of zeste mutations were observed only in a sensitized background in which pairing had been partially disrupted by chromosomal rearrangements (Gelbart and Wu 1982).

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Figure 6.—

Effects of zeste mutants on apterous transvection. (A) The gain-of function, hyperaggregating z¹ allele has little or no effect on ap^UGO35/ap^DG transvection, while the hypomorphic mutation, z^a, causes only a very modest decrease in the strength of ap^UGO35/ap^DG transvection (N ≥ 320). (B) z¹ suppresses the wing phenotype of ap^56f/ap^DG. z^a has little or no effect on the wings of ap^56f/ap^DG flies (N ≥ 114).

Transvection at the apterous locus:

Here we present evidence for transvection at the Drosophila apterous locus. While interallelic complementation at ap has been previously reported (Shtorch et al. 1995), the ap alleles were not molecularly characterized. Consequently, it was not clear whether the complementation between these alleles involved trans-regulatory interactions or occurred at the level of the mutant ap gene products. We have observed trans-regulatory interactions with several different classes of ap mutations.

The first type is the transvection seen in trans combinations between mutations that disrupt enhancers and mutations that disrupt the promoter. At the ap locus, this is illustrated by the ap^DG/ap^UGO35 combination (Figure 4, B, F, and G). Interestingly, the transvection observed between ap^DG and ap^UGO35 is sufficient to express ap at or near wild-type levels, as >90% of the wings are completely wild type. ap mutants are recessive, so there is likely a range of ap activity that is sufficient to produce wild-type wings (on the basis of the haplo-sufficiency of ap and the fact that the ap^2xE allele generated in parallel to ap^DG, which has a duplication of the wing enhancer, has wild-type wings, this range is likely to extend from at least 0.5 to 2 times normal levels).

It is unknown to what extent Dipterans have learned to exploit this interesting feature of their genomes for normal gene regulation. For example, it is unlikely that trans regulation occurs at the endogenous y locus in wild-type flies, as the enhancers appear to be strongly tethered in cis by the promoter. Instead, trans regulation is observed only at y when the enhancers are freed by deletion of the cis promoter (Morris et al. 1999a,b, 2004; Lee and Wu 2006). ap is clearly different from y in this respect as we also observe relatively strong trans regulation when the enhancer deletion, ap^DG, is combined with presumed ap-coding region mutations that are likely to retain an intact promoter (Figure 5C). Since the suppression of these coding region mutants by ap^DG is not as strong as that observed with the promoter deletion ap^UGO35, cis interactions between the upstream wing enhancer and the promoter of the mutant gene must compete with the ap^DG promoter in trans.

The second type of trans-regulatory interaction observed at ap is the transvection effects observed with boundary elements. We identified two different boundary insertions in the ap regulatory region. ap^MM-Mcp is an insertion of the Mcp-containing Flipper 2 transposon 403 bp upstream of the ap transcriptional start site between the wing enhancer and the ap promoter (Figure 1A). Although the Mcp element in this transgene contains both a boundary element and a PRE, our results indicate that the wing defects seen in homozygous or hemizygous ap^MM-Mcp flies are due to the enhancer-blocking activity of the boundary and not due to silencing by the Mcp PRE (Figure 1, B, F, and G; Figure 2, A and B). In the absence of an Mcp boundary insertion that lacks the PRE, the possibility remains that the Mcp PRE contributes to the ap wing phenotype. However, if this is the case, it is likely that the role of the PRE is a modulatory one, as the bxd PRE alone is not sufficient to cause wing defects (Figure 1B). ap^f00451 is a su(Hw)-containing piggyBac element and is also inserted between ap enhancer elements and the ap promoter (Figure 3).

One version of this boundary-element-induced transvection is that seen in the interallelic complementation between the boundary insertions and the ap promoter deletion, ap^UGO35. This trans-regulatory interaction is observed with both the Mcp and su(Hw) elements. The Mcp insert, ap^MM-Mcp, has a strong ap wing phenotype, but when it is combined with the promoter deletion, ap^UGO35, the wing defects are partially suppressed (Figure 2, A and C). The fact that full suppression is not observed in this combination, while it is observed when the enhancer deletion is combined with the promoter deletion, indicates that the Mcp element must be capable of partially blocking trans interactions between the ap^UGO35 wing enhancers and the ap^MM-Mcp promoter. This suggestion is substantiated by a comparison of the wing phenotypes in combinations between ap^UG035 and the enhancer deletion with (ap^DG-Mcp) and without (ap^DG) the Mcp element. While nearly full suppression is observed in the latter case, the suppression of the wing defects in ap^DG-Mcp/ap^UGO35 flies is comparatively modest (Figure 4, A, B, E, and F). This difference can be attributed to the ability of the Mcp element to block the ap enhancers in trans from activating the ap promoter in cis to the boundary. On the other hand, a comparison of the wing phenotype of the ap^DG-Mcp/ap^UGO35 trans combination (Figure 4A) with flies that are either hemizygous or homozygous for the Mcp insertion, ap^MM-Mcp (Figure 2A), reveals that the enhancer-blocking activity of this boundary element is stronger when the enhancer and promoter are in cis than when they are in a trans configuration.

The other version of boundary-element-induced transvection that we observed is the trans combination between the boundary insertions and the ap wing-enhancer deletion, ap^DG. This combination was tested for the Mcp and su(Hw) inserts and in both cases the wing phenotype of the enhancer deletion was suppressed (Figure 8, B, D, and E). Since the extent of suppression in both cases is considerably less than seen when the enhancer deletion ap^DG is combined with the promoter deletion ap^UGO35, it would appear that the boundary in cis to the enhancer is able to partially block its interactions with the ap promoter in trans. As noted above, the converse is also true: boundary elements in trans to the enhancer are able to partially block interactions with the ap promoter in cis.

Since these results demonstrate that the Mcp and su(Hw) boundaries can act not only in cis but also in trans, one might predict either that no interallelic complementation would be observed when two different boundary inserts are combined or that the phenotype would actually become even stronger because of the ability of boundaries to inhibit regulatory interactions in trans. Surprisingly, however, neither of these expectations holds. Instead, flies trans-heterozygous for the Mcp insert ap^MM-Mcp, and the su(Hw) insert ap^f00451 have completely wild-type wings (Figure 7, A–C). One mechanism that could account for this unexpected result is insulator bypass. Studies on the su(Hw) insulator have shown that enhancer-blocking activity is neutralized when there are two copies of this element in tandem between the enhancer and the promoter (Cai and Shen 2001; Muravyova et al. 2001). While bypass is thought to involve su(Hw)-pairing interactions, other insulators, including Mcp, can be substituted for one of the two su(Hw) elements (Melnikova et al. 2004). A strong prediction of the insulator bypass model is that interallelic complementation should also be observed when the su(Hw) element in ap^f00451 is in trans to the enhancer deletion that retains an intact Mcp element, ap^DG-Mcp. However, this is not the case as the wing phenotype of ap^DG-Mcp/ap^f00451 trans-heterozygotes is the same as that of ap^f00451 alone (Figure 8, A and C). This result indicates that the Mcp element is able to prevent trans activation of the ap promoter in cis by the wing enhancers on the ap^f00451 chromosome. The ability to block enhancers on the trans chromosome from contacting the promoter in cis to a boundary element was also observed when ap^MM-Mcp is combined with the Mcp-containing enhancer deletion ap^DG-Mcp (Figure 8A).

Thus, the interallelic complementation observed in ap^MM-Mcp/ap^f00451 flies is not likely to be an instance of insulator bypass. Instead, it seems that the additive effects of the unblocked, ap proximal portion of the ap^f00451 enhancer and trans activation by the enhancer on the ap^MM-Mcp chromosome (similar to that observed in Figure 8, B, D, and E) can account for the wild-type wings of ap^MM-Mcp/ap^f00451 flies (Figure 9A).

We thank Pam Geyer, Steven Cohen, Jumin Zhou, and Christian Sigrist for providing plasmids and Steven Cohen, Victor Corces, Francois Karch, the Bloomington Stock Center, and the Harvard–Exelixis stock collection for providing flies used in this study. We also thank Girish Deshpande, Greg Shanower, Tsutomu Aoki, and the Schedl lab for helpful discussions and Girish Deshpande and Martha Klovstad for comments on the manuscript. D.G. was supported by a Predoctoral Fellowship from the New Jersey Commission on Cancer Research. P.S., V.P., and M.A. would like to acknowledge support from the National Institutes of Health, the Kantons of Basel-Land and Basel-Stadt, and the Swiss National Science Foundation.