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Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences Are Involved in Anchoring Distant cis-Regulatory Regions to the Promoter
László Siposa, József Mihálya,b, François Karchb, Paul Schedlc, János Gausza, and Henrik Gyurkovicsaa Hungarian Academy of Sciences, Biological Research Center, Institute of Genetics, H-6701 Szeged, Hungary,
b University of Geneva, Department of Zoology and Animal Biology, CH-1211 Geneva 4, Switzerland
c Department of Biology, Princeton University, Princeton, New Jersey 08544
Corresponding author: Henrik Gyurkovics, Hungarian Academy of Sciences, Biological Research Center, Institute of Genetics, P. O. B. 521, H-6701 Szeged, Hungary, henrik{at}everx.szbk.u-szeged.hu (E-mail).
Communicating editor: T. SCHÜPBACH
| ABSTRACT |
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The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 1014) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-regulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the regulation of Abd-B are discussed. In addition, we present evidence that the tenacity of trans-interactions in the Abd-B gene may vary, depending upon the tissue and stage of development.
IN eukaryotes, gene activity can be controlled by extensive regulatory regions that are located many kilobases away from the promoter. In the most widely accepted model for such long-distance interactions, the intervening sequences between the regulatory regions and the promoter are thought to "loop out" (![]()
One model system for studying the factors governing such long-distance regulatory interactions is provided by the phenomenon of transvection in Drosophila. Transvection, first described by ![]()
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The Ubx gene is a part of the homeotic bithorax complex (BX-C). Although BX-C contains only two other homeotic genes, abdominal-A (abd-A) and Abdominal-B (Abd-B) (![]()
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; ZAVORTNIK and SAKONJU 1989), corresponding to the Abd-B r subfunction (![]()
RNA species in PS14 and PS15.
Recently, an unusual type of trans-regulation has been described for the Abd-B class A transcription unit and its regulatory regions (![]()
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| MATERIALS AND METHODS |
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General procedures:
Fly stocks were maintained on standard yeast-cornmeal medium. Crosses were performed at 25° en masse, unless otherwise noted. Unless described in this article, all genetic variants used are described in the following references: iab-7MX2, Abd-BD14, Abd-BD16, Df(3R)C4 (![]()
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2-3 (![]()
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Immunohistochemical staining of embryos:
Embryos collected from 17-hr egglays were dechorionated, fixed and devitellinized according to the procedure of ![]()
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Chromosomes generated by recombination:
McpB116iab-7Sz Abd-BD16:
The synthesis of this chromosome was done in three consecutive steps. First, we generated the chromosome McpB116 iab-7Sz by recombination. Out of ~29,000 male progeny of the cross between McpB116/iab-7Sz virgins and Oregon-R males, we identified two flies with darkly pigmented A4 [due to the dominant mutation, Miscadastral pigmentation (Mcp)] and an additional rudimentary seventh tergite (iab-7-). In the second step, Microcephalus (Mc) (a dominant mutation resulting in a strong reduction of the head capsule) was recombined onto the McpB116 iab-7Sz chromosome. McpB116iab-7Sz/Fab-7Mc virgins were mated with Oregon-R males. Among ~35,000 F1 males, we found three recombinants showing Mcp, iab-7 and Mc, but no Fab-7 phenotype. Finally, we isolated recombinants between the chromosomes McpB116 iab-7Sz Mc and Abd-BD16. (Mcp and Mc served as flanking markers to detect recombination events.) Dp(3;1)bxd111/+; Abd-BD16/McpB116 iab-7Sz Mc virgins were allowed to mate with Oregon-R males and their male progeny (~90,000) were scored for the presence of Mcp and the absence of Mc phenotype. Of the 39 such males found, eight were sterile and 23 did not carry Abd-BD16 [based on complementation with Df(3R)P9]. The remaining eight lines carrying the Abd-BD16 were tested for their ability to suppress the Fab-7 phenotype. Although all of them showed suppression, they fell into two discrete categories: five of them had an A6 tergite larger than the thin A7 (similar to the phenotype of Fab-7/Abd-BD16), whereas in the remaining three these tergites were equally large both in A6 and in A7. We concluded that the first group carried McpB116 only, and the second group had both McpB116 and iab-7Sz. We confirmed the presence of both lesions by Southern blot analysis. The same protocol was followed in the generation of recombinant chromosomes that contained Abd-BD14 instead of Abd-BD16.
Fab-7 Abd-BD16:
Two apparently contradictory observations prompted us to study transvection in the Abd-B domain. First, we found that (when the homologues are paired) an Abd-B point mutation trans to the Fab-7 mutation suppresses the Fab-7 gain-of-function phenotype so that the A6 tergite in males is larger than the A7 tergite (trans-suppression, described in detail in the RESULTS section). Second, we noticed in an earlier study (![]()
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R5LR7R:
For the construction of this chromosome, we took advantage of the fact that the desired product of recombination between the two inversions In(3R)iab-7R7 and In(3R)iab-7R5 should contain a deletion in the region 87C1-2;87D1-4, including the karmoisin (kar) locus at 87C8 (![]()
Isolation of rearrangements that eliminate trans-suppression:
We induced chromosomal rearrangements on the Fab-7 chromosome by irradiating Fab-7/Fab-7 homozygous males with X rays (4000 rads; 1000 rads/min, 0.5-mm Al filter) and crossing them to Abd-BD16/TM6B Tb virgins. After 6 days the parents were discarded. Among the male progeny (about 3000) we identified 12 flies in which trans-suppression was weaker or no longer visible. We then tested these new rearrangements over the Cbx1Ubx1 chromosomes. All but one showed significant reduction of the weak dominant wing phenotype generated by the Cbx1 mutation through the misexpression of Ubx+ on the homologous chromosome. Cytological examination of the exceptional case revealed a breakpoint at the BX-C, 89E1,2. By complementation analysis, we showed that the breakpoint inactivated the iab-4 cis-regulatory region.
We isolated transvection-disrupting rearrangements on the Fab-7 Abd-BD16 chromosome in a similar way. Irradiated Fab-7 Abd-BD16/TM6C Sb males were crossed to Cbx1Ubx1/TM1 females. Out of ~1500 F1 flies we isolated five individuals showing significantly reduced or no Cbx phenotype. Cytological examination of the two mutations that completely eliminated the Cbx phenotype, TSR-11A Fab-7 Abd-BD16 and TSR-59A Fab-7 Abd-BD16, showed the presence of the rearrangement T(3;4)89A-B;102A in the first, and Df(3R)88D;89D + Tp(3;2)89E;98C;39A in the second mutation.
Isolation of new deletions in the Abd-B gene by P-element remobilization:
In order to generate deletions that remove sequences around the promoter of the Abd-B class A transcription unit, the P-element insertion UC21-10,1-d, localized 253 bp upstream of the proximal Abd-B promoter (![]()
2-3. First, the Fab-7 mutation was recombined onto the chromosome carrying the insert. [In contrast to the original chromosome, these recombinants survive as adult homozygotes, suggesting that the reported lethality (![]()
2-3 males and Dp(3;1)bxd111/Dp(3;1)bxd111; ry506Abd-BD16/ry506 Abd-BD16 virgins. In contrast, all nine putative 5' deletions showed some degree of transformation of A6 toward A7 (Fab-7 phenotype) in trans to wild-type chromosomes. This phenotype allowed us to separate the chromosomes carrying the new derivatives of the insertional mutation from the ry506 Abd-BD16 chromosome and to establish stocks. Two of them, Abd-BPSz1 and Abd-BPSz2, showed significant complementation over iab-7Sz, i.e., the size of A7 was reduced compared to Abd-BD16/iab-7Sz,. Southern analysis detected a deletion of 10.8 kb between map positions +155.6156.8 and +165.6166.6 (3.2 kb downstream and 7.6 kb upstream of the site of the original insertion) in Abd-BPSz1, and a 5.5-kb deletion between map positions +155.6156.8 and +160.2161.3 (3.2 kb downstream and 2.3 kb upstream of the site of the original insertion) in Abd-BPSz2.
| RESULTS |
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In adult wild-type males the program specifying the development of the A7 abdominal segment does not produce a visible tergite or sternite (Figure 1A). This developmental program depends on the normal functioning of the iab-7 cis-regulatory domain that is responsible for generating the appropriate level of Abd-B RNA/protein expression in PS12/A7. When iab-7 is deleted on both homologues, Abd-B expression in PS12/A7 comes under the control of the iab-6 cis-regulatory domain, resulting in the transformation of PS12/A7 into a copy of PS11/A6 (![]()
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A phenotype exactly the opposite of that produced by iab-7 mutations is observed for the gain-of-function mutation Fab-7; PS11/A6 is transformed into PS12/A7. This is due to the ectopic activation of iab-7 in PS11/A6. As a consequence, the number of visible segments in Fab-7 homozygous males is reduced from six to five (![]()
The incomplete transformation of PS11/A6 in Fab-7/+ males could be attributed to a haplo-insufficiency for iab-7. In Fab-7/+ males, the iab-7 cis-regulatory domain on the Fab-7-containing homologue is active in PS11/A6, whereas the iab-7 cis-regulatory domain on the wild-type homologue is not. If regulation only occurs in cis, Abd-B expression in PS11/A6 should be driven by interactions between the iab-7 cis-regulatory domain and the Abd-B gene only on the Fab-7 homologue. Such interactions should not occur on the wild-type homologue in PS11/A6 because iab-7 is inactive. This interpretation is supported by the phenotype of Fab-7/iab-7Sz mutant animals. In this genotype, only a single copy of the iab-7 cis-regulatory domain should be available to drive Abd-B expression, not only in A6 and but also in A7. In males carrying this mutant combination, both PS11/A6 and PS12/A7 would be expected to assume an identity in between that of PS11/A6 and PS12/A7. This is the case. Moreover, as illustrated in Figure 1D and Figure 2, segments A6 and A7 in the Fab-7/iab-7Sz males closely resemble the A7 segment found in iab-7-/+ (Figure 1B) males.
Taken together, these results would seem to suggest that, if the rest of the Abd-B domain remains unchanged, the phenotype/identity of PS12/A7 (or PS11/A6 in the Fab-7 mutant) directly reflects the number of active iab-7 cis-regulatory domains.
Fab-7 is suppressed by Abd-B point mutations in trans:
We next examined the effects of combining Fab-7 with an Abd-B point mutation, Abd-BD16. If our hypothesis that the identity of PS11/A6 and PS12/A7 depends upon the number of iab-7 domains that are active in these two parasegments is correct, then Fab-7/Abd-BD16 males would be expected to have the same intermediate phenotype in A6 as was observed in Fab-7/iab-7Sz males. However, as illustrated in Figure 1E and Figure 2, this is not the case. Although A7 has the same phenotype in the two mutant combinations, the phenotype of segment A6 in Fab-7/Abd-BD16 males differs from that in Fab-7/iab-7Sz males. The A6 tergite in the Fab-7/Abd-BD16 genotype is enlarged relative to the A7 tergite, and more closely resembles the wild-type A6 segment. This unexpected finding suggests that, unlike iab-7Sz, the Abd-BD16 mutation partially suppresses the gain-of-function phenotype of Fab-7 in A6. This partial suppression is not due to some unusual properties of the Abd-BD16; precisely the same A6 phenotype was observed when other Abd-B point mutations (Abd-BS1, Abd-BS4; ![]()
These observations could mean that the activity of Abd-B is "more haplo-insufficient" in A6 than in A7. If this were true, we would expect to observe a similar suppression of the Fab-7 phenotype in A6 by the deletion Df(3R)P9, which removes the entire BX-C, including the Abd-B gene. However, as was observed for the Fab-7/iab-7Sz combination, segments A6 and A7 in Fab-7/Df(3R)P9 males show the same intermediate A6-A7 phenotype (see Figure 1F). Similarly, the combination of Fab-7 with a deletion, Df(3R)R59, which removes both iab-6 and iab-7 but not the Abd-B gene, has the same phenotype as Fab-7/iab-7Sz (data not shown), indicating that the functioning of iab-6 in trans is irrelevant for the phenotype of Fab-7 in A6.
Chromosomal rearrangements eliminate the effect of the Abd-B point mutations:
The suppression of the Fab-7 gain-of-function phenotype by Abd-B point mutations in trans implies that our original assumption, namely, that regulatory interactions only occur in cis, is incorrect. Instead, it suggests that some type of trans-regulatory interaction or "transvection" must also occur. In particular, a trans-regulatory interaction could potentially explain why the Fab-7 gain-of-function phenotype of Fab-7/Abd-B+ animals is stronger than that of Fab-7/Abd-B- animals; in the former case, the Abd-B genes in cis and in trans would produce functional products, whereas in the latter case, functional products would only be produced by the Abd-B in cis.
Transvection is classically tested by generating chromosomal rearrangements that disturb homologous pairing. For this purpose, we first screened for X-ray-induced mutations on the Fab-7 chromosome, which reduced or eliminated the suppression of the Fab-7 gain-of-function phenotype by Abd-BD16, that is, for Fab-7*/Abd-BD16 males in which the A6 tergite more closely resembled the A7 tergite. [We used Abd-BD16 as a representative allele in this and subsequent experiments because it is a molecularly characterized mutation that does not code for a detectable protein (![]()
Chromosomal rearrangements that interfere with the postulated transvection between Fab-7 and Abd-B might also be expected to disrupt pairing-dependent interactions elsewhere in BX-C. A well-known example of a pairing-dependent regulatory interaction in BX-C is the wing transformation induced by the Cbx1Ubx1 chromosome when it is paired with a wild-type copy of BX-C (![]()
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As expected from the suppression of the Cbx1 phenotype, cytological examination of these 11 Fab-7* chromosomes revealed that they all had rearrangements affecting the right arm of the third chromosome. The position of the breakpoints for the 11 Fab-7* chromosomes that suppress the Cbx1 phenotype (TSR-1Sz,Fab-7 to TSR-11Sz,Fab-7) are listed in Table 1. All 11 of them have one breakpoint between BX-C and the centromere, and a second breakpoint outside of this region. This arrangement of breakpoints follows the rules established by ![]()
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These results argue that the suppression of Fab-7 gain-of-function phenotype by Abd-B point mutations is likely to be due to a nonproductive trans-regulation. When a wild-type gene is present on both homologues (Fab-7/+), both cis- and trans-regulatory interactions can contribute to the level of ABD-B protein that is ultimately expressed in PS11/A6. Although these same cis- and trans-interactions would also occur in Fab-7/Abd-BD16 flies, functional Abd-B protein would only be produced by the Abd-B gene on the Fab-7 chromosome. It should be noted that this model requires that the regulatory capacity of the ectopically activated iab-7 domain in PS11/A6 is limiting; otherwise, the presence of a competing Abd-B point mutant in trans to the Fab-7 chromosome would have no phenotypic consequences. This suppositionthat the regulatory capacity of iab-7 is limitingseems to be correct, because the phenotype of PS11/A6, when the Abd-B gene in trans is either removed entirely (as in Fab-7/Df(3R)P9) or no longer available for pairing (as in TSR,Fab-7/Abd-BD16), is indistinguishable from Fab-7/+.
Competition between the iab-7 cis-regulatory domains:
In the experiments described in the previous section, nonproductive trans-interaction between the iab-7 regulatory domain on the Fab-7 homologue and the Abd-B point mutation on the other homologue suppressed the gain-of-function phenotype in A6. We speculated that the trans-interactions between iab-7 and the mutant Abd-B gene were permitted in A6 because the iab-7 regulatory domain on the homologue containing the mutant Abd-B gene is not active in this segment (see ![]()
If this model is correct, cis-competition in segment A7 should be eliminated by deleting the iab-7 regulatory domain on the Abd-BD16-containing chromosome. The lack of competition from the iab-7 domain in cis should result in an increase in the frequency or strength of trans-interactions between the iab-7 domain on the Fab-7 chromosome and the mutant Abd-B gene on the other homologue.
To test this prediction, we recombined an iab-7 deletion mutant, iab-7Sz, onto the Abd-BD16 chromosome. The phenotype of +/iab-7SzAbd-BD16 and Fab-7/iab-7SzAbd-BD16 males is shown in Figure 1H and Figure 1G, respectively. Two findings are of interest.
First, the gain-of-function phenotype of Fab-7 in A6 is still suppressed in Fab-7/iab-7SzAbd-BD16 males (Figure 1G), and this segment closely resembles the A6 segment found in Fab-7/Abd-BD16 males (Figure 1E). The similarity between these two mutant combinations would argue that the iab-7 cis-regulatory domain on the Abd-BD16 chromosome does not contribute to the suppression of the Fab-7 phenotype in A6 and would be consistent with the idea that the iab-7 cis-regulatory domain is normally inactive in this segment (![]()
These findings indicate that trans-regulatory interactions in segment A7 have no phenotypic consequences if an iab-7 domain is present on the chromosome containing the mutant Abd-B gene. To demonstrate that this domain must not only be present but also active, we recombined Fab-7 with the Abd-B point mutation Abd-BD16 and then examined the phenotype of Fab-7/Fab-7Abd-BD16 males. In these animals, both copies of iab-7 will be ectopically activated in A6. If we are correct in assuming that the regulatory domain in cis must be active in order to reduce trans-regulatory interactions with the mutant Abd-B gene, then the phenotype of A6 and A7 in these animals should be identical and resemble the A7 segment in Fab-7/Abd-BD16 animals. Indeed, this is the case (see Figure 2).
Two different (but not mutually exclusive) hypotheses could explain the phenotypic difference between the segments in which one vs. two iab-7 regions are active, e.g., the A7 of Fab/iab-7Sz Abd-BD16 and Fab-7/Abd-BD16. The first postulates that the iab-7 regulatory domains engage not only in cis- but also in trans-regulatory interactions (indicated by dotted arrows in Figure 2). In this case, the resulting phenotype depends upon the sum of the interactions contributed by either one or two active iab-7 domains. In the second, cis-interactions between the iab-7 regulatory domain and the Abd-B gene on the same homologue would, because of competition, tend to reduce or suppress trans-interactions between this Abd-B gene and the iab-7 regulatory domain on the other homologue. When there is no competing active regulatory domain in cis, the Abd-B gene will be more readily available to engage in trans-interactions. It is not possible to distinguish between these two models with the genetic tools presently available; however, we favor the second model, as our analysis of the effects of promoter deletions (see below) suggests that the balance between cis- and trans-interactions can be shifted by competition.
Mutations that delete sequences near the 5' end of the Abd-B gene enhance trans-regulation:
The ability of the iab-7 regulatory domain to interact with the Abd-B genes in cis and in trans is not equal. If the cis- and trans-interactions were symmetrical, the phenotype of Fab-7+/+Abd-B and Fab-7Abd-B/++ would be identical. However, this is clearly not the case. In contrast to the modest but detectable transformation of A6 toward A7 in Fab-7/Abd-BD16 flies, we see no trace of an A6 to A7 transformation in Fab-7Abd-BD16/+ flies. Similarly, Abd-BD16 does not complement iab-7Sz to any detectable degree in A7 (Figure 6A).
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We reasoned that it might be possible to strengthen the interaction of iab-7 with the trans copy of Abd-B by weakening its interactions with the Abd-B gene in cis. Because the regulatory interactions of iab-7 with the Abd-B gene are likely to be mediated, at least in part, by sequences in the vicinity of the Abd-B promoter, we thought that it might be possible to weaken the cis-interactions by substituting Abd-B point mutants with an Abd-B 5' deletion mutant, Abd-BD14 (m-; ![]()
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To rule out the possibility that this weak transformation is due to a "leakiness" of the Abd-BD14 mutation, we examined the phenotype of Fab-7Abd-BD14/Df(3R)P9 hemizygotes and Fab-7Abd-BD14/Fab-7Abd-BD14 homozygotes. In neither case did we detect transformation of A6 into A7 (not shown). In fact, it appears that the weak transformation observed in Fab-7Abd-BD14/+ animals requires that the wild-type copy of the Abd-B gene in trans can pair. Thus, transformation is not detectable when pairing is efficiently disrupted (Figure 3B). Conversely, trans-interaction can be detected in both Fab-7Abd-BD14/iab-7Sz and Fab-7Abd-BD14/Df(3R)R59 flies (not shown), where the trans copy of the iab-7 regulatory domain is deleted but the Abd-B gene is intact.
Other lines of evidence also suggest that the Abd-BD14 promoter deletion mutant is a weaker competitor for interactions with iab-7, either in cis or in trans, than a structurally wild-type Abd-B gene. First, the trans-suppression of the Fab-7 gain-of-function phenotype in segment A6 of Abd-BD14/Fab-7 males is significantly weaker (but still detectable) than in the combination of Fab-7 with any of the Abd-B point mutants (compare Figure 4B and a). Second, the haplo-insufficient phenotype of Abd-BD14/+ in A7 is clearly weaker than that of Abd-B point mutations (compare Figure 4B and a), which suggests a hyperactivation of the wild-type Abd-B gene in trans. These differences are pairing dependent, because the phenotypes of Abd-BD14/TSR,Fab-7 (Figure 4C) and Abd-BD16/TSR,Fab-7 flies are indistinguishable (not shown).
Isolation of new Abd-B mutations which enhance trans-regulation by iab-7:
The observation that the small Abd-BD14 deletion enhances iab-7 trans-regulation points to sequences in the vicinity of the proximal Abd-B promoter as potential targets for cis- (and trans-) regulatory interactions. However, the finding that Abd-BD14 still suppresses Fab-7 in trans to some extent (compare Figure 4B and Figure C) suggests that additional sequences may also be relevant for trans-regulation.
To further characterize sequences from the Abd-B gene that contribute to these regulatory interactions we took advantage of a hypomorphic Abd-B mutation, UC21-10,1-d, which has a P-transposon at position +159 on the molecular map (![]()
To identify sequences that contribute to cis- (and trans-) regulatory interactions, we mobilized the UC21-10,1-d transposon on the Fab-7UC21-10,1-d chromosome and then selected for excision events that eliminated the remaining Abd-B m activity (see MATERIALS AND METHODS). We then tested these new Abd-B alleles for their ability to enhance trans-regulation of a wild-type Abd-B. Out of nine new Abd-B alleles, we identified two mutations (Abd-BPSz1 and Abd-BPSz2) that enhanced the Fab-7 gain-of-function phenotype in A6 when trans to a wild-type copy of BX-C more strongly than Abd-BD14. Genomic Southern blotting revealed that Abd-BPSz1 has a 10.8-kb deletion between map position +155.6156.8 and +165.6166.6, and Abd-BPSz2 carries a smaller deletion of about 5.5-kb from +155.6156.8 to +160.2161.3 (Figure 5).
Other 5' deficiencies also enhance trans-regulation:
When we compared the transformation of A6 into A7 in Fab-7 Abd-B-/+ flies, it appeared that the strength of the trans-activation of the wild-type Abd-B gene is greatest in the largest promoter deletion, that is, Fab-7Abd-BPSz1/+ > Fab-7Abd-BPSz2/+ > Fab-7Abd-BD14/+ > Fab-7Abd-BD16/+. Interestingly, precisely the same relationship is observed for the complementation of the iab-7 deletion, iab-7Sz, in A7. Thus, the strongest complementation is observed in Fab-7Abd-BPSz1/iab-7Sz flies, which is followed by Fab-7Abd-BPSz2/iab-7Sz, Fab-7Abd-BD14/iab-7Sz, and finally, Fab-7Abd-BD16/iab-7Sz. The similarity between these two assays suggested that we could measure the ability of any Abd-B mutation to promote trans-regulation by examining its complementation of iab-7Sz in A7. Using this assay, we tested the trans-regulating ability of three previously isolated deficiencies, Df(3R)C4 (![]()
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The approximate limits of the complementing deletions are shown in Figure 5. These deletions can be ordered in increasing strength of iab-7Sz complementation as follows: Abd-BD14 < Abd-BPSz2 < Abd-BRD18
Abd-BPSz1. From this data it would appear that trans-regulation is strongest in the case of deletions that remove the largest region of Abd-B DNA on the 5' side of the Abd-B gene (compare Figure 6B and Figure C). It is also clear that deficiencies that strongly enhance trans-regulation extend well beyond the region thought to correspond to the proximal Abd-B promoter (![]()
We note that unlike Abd-B "point mutations," the Abd-BPSz2, Abd-BRD18 or Abd-BPSz1 deletions do not show any sign of an A7 to A6 transformation when they are trans to a wild-type BX-C. This difference presumably reflects the ability of these large promoter deletions to enhance trans-regulatory interactions with the wild-type Abd-B gene. In fact, in the case of these larger deletions, the hyperactivation of the wild-type Abd-B gene in trans is even stronger than that observed in the case of the smaller promoter deletion, Abd-BD14, because a weak A7 to A6 transformation (the presence of at least a single bristle in tergite A7; Figure 4B) is observed in Abd-BD14/+ males. However, these promoter deletions do show the expected haplo-insufficient phenotype when they are combined with a chromosome like TSR, Fab-7 that disrupts pairing. In this case, their phenotype is indistinguishable from that of TSR,Fab-7/Abd-BD14 (see Figure 4C).
Df(3R)U110 is an unusual case. It is the largest of the Abd-B deficiencies that still retains iab-7, and it extends from near the 3' end of Abd-B into the 90A region. As expected from the large size of this deficiency, it exhibits by far the strongest enhancement of trans-regulation when in trans to iab-7Sz. However, unlike any of the other deficiencies, the extent of complementation by the trans copy of iab-7 varies from hemitergite to hemitergite and sometimes appears to be clonal even within a hemitergite (Figure 6D). Df(3R)U110 also differs from the other Abd-B deficiencies in that it fails to complement the loss-of-function iab-7 phenotype of Df(3R)R59, a deficiency that extends proximally from the 3' end of the Abd-B gene to the bxd region. The only sign of some complementation in Df(3R)U110/Df(3R)R59 males is the presence of black pigmentation in tergite A6 and A7, a phenotype corresponding to the result of the pairing-insensitive, long-range interaction described by ![]()
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The most likely explanation for the unusual behavior of Df(3R)U110 is illustrated in Figure 7. In the case of nonoverlapping deficiency combinations such as Abd-BRD18/iab-7Sz (Figure 7A), the iab-7 and Abd-B DNA segments on the two homologues loop out, but remain in relatively close proximity. This also holds for the overlapping deficiency combination Abd-BRD18/Df(3R)R59 (see Figure 7B). In contrast, the remaining iab-7 and Abd-B DNA segments in the Df(3R)U110/Df(3R)R59 are located on opposite sides of the large unpaired deficiency loops (see Figure 7C), and this distance (which to a first approximation corresponds to the size of the smaller deficiency) may prevent effective trans-regulation. In the case of the Df(3R)U110/iab-7Sz, the iab-7 deficiency is quite small, and the remaining iab-7 and Abd-B DNA segments would still be in relatively close proximity. (The hypothetical chromosome structure of Df(3R)U110/iab-7Sz is essentially the same as that of Abd-BRD18/iab-7Sz, shown in Figure 7A.) Thus, the difference between the phenotype of Df(3R)U110/iab-7Sz and that of Df(3R)U110/Df(3R)R59 would suggest that proximity has a strong influence in the restoration of A7 identity by trans-regulation.
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Transvection in the Abd-B domain is not zeste-dependent:
Because most of the previously documented cases of transvection are dependent on the presence of a wild-type copy of the X-linked gene zeste (![]()
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Is transvection tissue or stage specific?
We wondered whether iab-7 is able to trans-regulate the Abd-B gene in tissues besides those giving rise to the adult cuticle. To investigate this question, we used immunostaining to examine the Abd-B expression in embryos of different genetic backgrounds.
The various control embryos gave the expected patterns of Abd-B protein expression described by others (![]()
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We next examined the Abd-B expression pattern in Fab-7Abd-BD16/Df(3R)R59 and Fab-7Abd-BD14/Df(3R)R59 embryos. On the basis of regulatory interactions inferred from the phenotype of the adult cuticle, we anticipated that the Abd-B gene on the R59 homologue would be trans-activated in the embryos carrying the Abd-BD14 deletion mutant, but not in the embryos carrying the Abd-BD16 point mutation. For Fab-7Abd-BD14/Df(3R)R59 embryos, we found the same levels of ABD-B protein in the CNS of PS11 and PS12 (see Figure 8D). This is the result expected if the iab-7 regulatory domain on the Fab-7 chromosome is able to trans-regulate the Abd-B gene on the R59 homologue in PS11 and PS12. Thus, for this mutant combination, trans-regulation is the same in the embryonic CNS as it is in the adult. Surprisingly, although we were unable to detect trans-regulation of the Abd-B gene on the R59 homologue in adult Fab-7Abd-BD16/Df(3R)R59 animals, trans-regulation could be detected in the embryonic CNS. As in Fab-7Abd-BD14/Df(3R)R59 embryos, we found the same levels of ABD-B protein in the CNS in PS11 and PS12 of Fab-7Abd-BD16/Df(3R)R59 embryos (not shown).
Although we found clear evidence for trans-regulatory interactions between the Fab-7Abd-B- and R59 homologues in the embryonic CNS, such interactions were not evident in other embryonic tissues. Thus, in animals of the same two genotypes, we were unable to detect ABD-B protein in either the ectoderm or mesoderm of PS11 and PS12. This was true even at the beginning of germ-band retraction, when Abd-B expression is most readily evident in wild-type embryos in these parasegments (not shown).
In some experiments, we saw faint Abd-B specific staining of CNS in PS10 of both Fab-7Abd-BD14/Df(3R)R59 and Fab-7Abd-BD16/Df(3R)R59 embryos. This observation suggested that iab-5 and iab-6 may also be able to trans-regulate the Abd-B gene on the R59 homologue. To test this possibility, we stained McpB116iab-7SzAbd-BD16/Df(3R)R59 embryos. The McpB116 mutation is a deletion that removes a putative boundary between iab-4 and iab-5, ectopically activating the PS10-specific iab-5 in PS9 (![]()
Long distance trans-regulatory interactions in the embryonic CNS:
The results described in the previous section indicate that trans-regulatory interactions in the Abd-B domain may be significantly stronger in the embryonic CNS than in the adult epidermis. If this were true, it might be possible to detect trans-regulation in the CNS of embryos carrying chromosomal rearrangements that substantially perturb pairing in BX-C and eliminate transvection in the cells giving rise to the adult epidermis. To investigate this possibility we examined Abd-B expression in the CNS of embryos heterozygous for either Fab-7Abd-BD14 or Fab-7Abd-BD16 and chromosomes that have rearrangements that disrupt Abd-B and Ubx trans-regulation in the adult. These rearrangements include TSR-11A, TSR-59A, iab-7MX2 (In(3LR)64A; 89A;89E; KARCH et al. 1985), iab-7164 (In(3LR)65;89E superimposed on In(3R)1000 with breaks in 81C and 90C; CELNIKER et al. 1990).
Even though these rearrangements suppress trans-regulatory interactions in the cells giving rise to the adult epidermis, they do not prevent trans-regulatory interactions in the embryonic CNS, and we observe equal levels of ABD-B protein in PS11 and PS12 (as would be expected if the iab-7 regulatory domain on the Fab-7Abd-B- chromosome is driving the expression of the Abd-B gene on the rearranged chromosome). The one exceptional case is the doubly rearranged iab-7164 chromosome. In both Fab-7Abd-BD16/iab-7164 and Fab-7Abd-BD14/iab-7164 embryos, the Abd-B antibody staining is weak and irregular (Figure 8H), suggesting that the configuration of the rearrangement in this particular chromosome interferes with trans-regulation even in the CNS.
Long-distance regulatory interactions in the embryonic CNS are suppressed by the uninterrupted pairing of homologues:
Our ability to detect long-distance trans-regulatory interactions in the CNS prompted us to investigate the parameters governing this phenomenon further. The chromosomal rearrangement In(3R)Fab-7iab-7R5 was isolated as a revertant of Fab-7. It has a distal breakpoint at position +152 on the molecular map in the BX-C, very close to the 3' end of the Abd-B m transcription unit. From its position, it seems likely that the break in BX-C may leave both the iab-7 regulatory domain and the Abd-B gene functionally intact. The proximal breakpoint is in the region 87C1-2,3.
Because iab-7 as well as iab-5 and iab-6 are separated by a large distance from the Abd-B gene in this inversion-bearing chromosome, these regulatory domains should be unable to contact the Abd-B gene, and the gene should not be expressed in PS10PS12 in the CNS. This is the case in embryos homozygous for the In(3R)Fab-7iab-7R5 inversion; we only see Abd-B staining in PS13PS14 (not shown). Surprisingly, however, a different result is obtained when In(3R)Fab-7iab-7R5 is combined with the deficiency chromosome Df(3R)P9 that removes the entire BX-C. In this combination, we detect a low but uniform level of Abd-B antibody staining in the CNS of PS11 and PS12 (Figure 8F).
From these results, it would appear that iab-7 is able to regulate a distantly located Abd-B gene on the same chromosome when the inversion is in trans to a BX-C deficiency, but not when it is homozygous. In the former case, two factors might facilitate this regulatory interaction. First, the loop formed by homologous pairing between the deficiency and the inversion chromosomes would bring the BX-C sequences on either side of the inversion breakpoints in close proximity. Second, there would be unpaired regions around the breakpoints of the inversion that at one end might expose the iab-7 regulatory domain and at the other end might expose the Abd-B gene and 5' flanking regions. In the latter case, the iab regulatory domains and the Abd-B gene would not be brought in close proximity when the two inversion chromosomes pair. Instead, these sequences would be separated by the length of the inversion. Additionally, the pairing of the BX-C regions across the inversion breakpoint would be uninterrupted.
We wondered whether unpaired DNA segments could by themselves facilitate long-distance interactions. To investigate this possibility, we combined In(3R)Fab-7iab-7R5 with another inversion, In(3R)Fab-7iab-7R7, which has its breakpoints in 87D1,2 and 89E3,4 (![]()
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