Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences Are Involved in Anchoring Distant cis-Regulatory Regions to the Promoter

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 10–14) 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" (PIRROTTA 1991 ). Although the looping model can account for long-distance interactions between regulatory elements and promoters, it also poses a problem. Most regulatory elements are rather promiscuous in their interactions and are capable of controlling the activity of many different promoters, irrespective of their origin. Thus, the looping model raises the question of how enhancers are able to distinguish their target promoter from the promoters of other nearby genes.

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 LEWIS 1954 in the Ultrabithorax (Ubx) locus, refers to a partial interallelic complementation that depends upon the pairing of homologous chromosomes. In the best-documented cases, such as in the yellow (GEYER et al. 1990 ) and the Ubx loci (MARTINEZ-LABORDA et al. 1992 ), transvection appears to involve trans-regulation, that is, a regulatory element on one of the homologues controls the promoter activity of the corresponding gene on the other, paired homologue. This unusual trans-interaction can be exploited to learn more about the mechanisms responsible for specifying conventional interactions between regulatory elements and promoters in cis.

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) (SANCHEZ-HERRERO et al. 1985 ; TIONG et al. 1985 ), these three genes assign proper identity to the third thoracic (T3) and all of the abdominal (A1–A9) segments, corresponding to parasegments (PS) 5 to PS14. This is achieved through the control exerted by PS-specific cis-regulatory elements on the individual genes of the complex. Thus, abx/bx and pbx/bxd elements regulate Ubx expression in PS5 and PS6 (BEACHY et al. 1985 ; HOGNESS et al. 1985 ; WHITE and WILCOX 1985 ), and iab-2, -3 and -4 elements regulate abd-A in PS7, PS8 and PS9, respectively (KARCH et al. 1990 ; MACIAS et al. 1990 ). iab-5 - iab-8 elements regulate the Abd-B class A transcription unit that corresponds to the Abd-B m function (CASANOVA et al. 1986 ) in PS10 through PS13 (CELNIKER et al. 1990 ; BOULET et al. 1991 ; SANCHEZ-HERRERO 1991 ). Specific regulatory elements that regulate the longer transcription units (class B, C and ; ZAVORTNIK and SAKONJU 1989), corresponding to the Abd-B r subfunction (CASANOVA et al. 1986 ), have not yet been identified, but they are expected to specify the expression pattern of the class B, C and 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 (HENDRICKSON and SAKONJU 1995 ; HOPMANN et al. 1995 ). In contrast to the classical transvection of LEWIS 1955 , this trans-regulation is extremely resistant to disruption of homologous pairing. In this article, we provide evidence that trans-interaction in the Abd-B gene may vary in its resistance to disruption of homologous pairing in different tissues. With the help of a dominant gain-of-function mutation, Fab-7, we show that transvection of a more regular type, closely resembling the classical case in the Ubx locus, can also be demonstrated in Abd-B in the adult stage. Detailed analysis of this transvection suggests the existence of a multicomponent tethering mechanism that may ensure cis-autonomy of the Abd-B domain within the BX-C. This mechanism involves an extensive region upstream of the Abd-B gene, which appears to be distinct from the sequences required for transcription initiation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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-7^MX2, Abd-B^D14, Abd-B^D16, Df(3R)C4 (KARCH et al. 1985 ); Abd-B^RD18, Df(3R)U110 (HOPMANN et al. 1995 ); Abd-B^S1, Abd-B^S4 (TIONG et al. 1985 ); Abd-B^R41, Fab-7, In(3R)Fab-7iab-7^R7, Df(3R)R59 (GYURKOVICS et al. 1990 ); iab-7¹⁶⁴ (CELNIKER et al. 1990 ); iab-7^Sz (GALLONI et al. 1993 ); Mcp^B116 (KARCH et al. 1994 ); TM3 Sb P(ry⁺)2-3 (REUTER et al. 1993 ); UC21-10,1-d (MCCALL et al. 1994 ); Cbx¹, Df(3R)P9, Dp(3;3)P5, In(3LR)TM1, In(3LR)TM3 Sb Ser, In(3LR)TM6B Tb, In(3LR)TM6C Sb, Mc, Tp(3;1)bxd¹¹¹, Ubx¹, z¹, z^a, z^op, z^v77h (LINDSLEY and ZIMM 1992 ). In(3R)Fab-7iab-7^R5 was isolated as an X-ray-induced revertant of Fab-7, and its breakpoint within the BX-C was cloned and determined by Southern analysis. Cytological analysis of polytene chromosomes was performed as described by ASHBURNER 1989 . Adult abdominal cuticles were mounted as described by DUNCAN 1982 , and Southern blot analyses were done as described by BENDER et al. 1983 .

Immunohistochemical staining of embryos:
Embryos collected from 17-hr egglays were dechorionated, fixed and devitellinized according to the procedure of MITCHISON and SEDAT 1983 , modified as described in KARCH et al. 1990 . We modified this procedure further as follows. The time of fixation was reduced to 12 min. Devitellinized embryos were washed and rehydrated in 1x PBT (1x PBT = 1x phosphate-buffered saline + 0.1% Triton X-100 + 0.1 bovine serum albumin, 1x phosphate-buffered saline = 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄; pH = 7.5). Embryos were incubated with primary antibody (monoclonal mouse-anti-ABD-B 1A2E9; CELNIKER et al. 1990 ) diluted in 1x PBT 1:1 on a rotating wheel overnight at 4°C. Antibody was removed, and embryos were washed six times with PBT for about 20 min on rotating wheel. Horseradish peroxidase-conjugated rabbit-anti-mouse secondary antibody (DAKO), diluted in prechilled (4°) PBT 1:200, was added and incubated with the embryos on a rotating wheel for 3 1/2 hr at 4°. These conditions reduced background staining without significant loss of specific staining. Embryos were then washed again as described above. After removing PBT, embryos were briefly rinsed twice in staining buffer (0.1 M citric acid, 0.05 M NH₄-acetate, pH = 5.7 adjusted with NH₄OH) and stained in the mixture of 980 µl buffer, 20 µl DAB solution (25 mg/ml stock solution, final cc 0.5 mg/ml) and 2 µl H₂O₂ (30% stock solution, final cc 0.06%). The reaction was stopped by dilution with PBT and embryos were rinsed six times with PBT. Embryos were stored in PBT with 0.05% NaN₃ and mounted in 9:1 glycerol:10 x PBT. For the dissection of CNS, we used tungsten needles.

Chromosomes generated by recombination:
Mcp^B116iab-7^Sz Abd-B^D16: The synthesis of this chromosome was done in three consecutive steps. First, we generated the chromosome Mcp^B116 iab-7^Sz by recombination. Out of ~29,000 male progeny of the cross between Mcp^B116/iab-7^Sz 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 Mcp^B116 iab-7^Sz chromosome. Mcp^B116iab-7^Sz/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 Mcp^B116 iab-7^Sz Mc and Abd-B^D16. (Mcp and Mc served as flanking markers to detect recombination events.) Dp(3;1)bxd¹¹¹/+; Abd-B^D16/Mcp^B116 iab-7^Sz 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-B^D16 [based on complementation with Df(3R)P9]. The remaining eight lines carrying the Abd-B^D16 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-B^D16), whereas in the remaining three these tergites were equally large both in A6 and in A7. We concluded that the first group carried Mcp^B116 only, and the second group had both Mcp^B116 and iab-7^Sz. 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-B^D14 instead of Abd-B^D16.

Fab-7 Abd-B^D16: 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 (GYURKOVICS et al. 1990 ) that when the Fab-7 mutation is present both in cis and in trans to an Abd-B mutation (Fab-7/Fab-7 Abd-B^R41) the A6 and A7 tergites are equally thin. We presumed that this phenotypic difference between the two types of mutant combinations was not due to some unusual properties of Abd-B^R41 (which is an Abd-B m^- mutation, GYURKOVICS et al. 1990 ), but to the presence of the Fab-7 mutation in cis to Abd-B^R41. To test if this is the case, we attempted to isolate recombinants carrying both Fab-7 and Abd-B^D16 (an Abd-B mr null mutation; BOULET et al. 1991 ) on the same chromosome, based on the assumption that Fab-7/Fab-7Abd-B^D16 flies should have a phenotype similar to Fab-7/Fab-7 Abd-B^R41. For this purpose, ry⁵⁰⁶Fab-7/Abd-B^D16 females were crossed to ry⁵⁰⁶Fab-7/ry⁵⁰⁶Fab-7 males, and the male progeny were scored for the presence of equally thin A6 and A7 tergites. Out of ~35,000 males, three such flies were found. After establishing stocks of these lines, the presence of the Fab-7 deletion was confirmed by Southern blot analysis and the presence of Abd-B^D16 by genetic complementation test in all three putative recombinants. Having established the usefulness of this method, we constructed double mutant combinations of Fab-7 with Abd-B^D14 and UC21-10,1-d in the same way.

R5^LR7^R: 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-7^R7 and In(3R)iab-7^R5 should contain a deletion in the region 87C1-2;87D1-4, including the karmoisin (kar) locus at 87C8 (GAUSZ et al. 1979 ). Tp(3;1)bxd¹¹¹/+ ; iab-7^R5/iab-7^R7 females were crossed to cu kar/cu kar males, and the progeny were scored for kar phenotype. Of 180 flies scored, we recovered seven kar individuals. Stocks of these putative recombinants were established and checked cytologically. All of them contained the inversion In(3R)87C1-2;89E3-4 and the deficiency Df(3R)87C1-2;87D1-4.

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-B^D16/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-B^D16 chromosome in a similar way. Irradiated Fab-7 Abd-B^D16/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-B^D16 and TSR-59A Fab-7 Abd-B^D16, 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 (MCCALL et al. 1994 ), was mobilized by introducing the transposase source P(ry⁺) 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 (MCCALL et al. 1994 ) should be due to second-site mutations located proximal to the Fab-7 mutation.] Fab-7UC21-10,1-d/+ flies show a moderate Fab-7 phenotype that is strongly reduced if these chromosomes are over an Abd-B null mutation, suggesting that the Fab-7 phenotype originates mainly from an interaction with the paired wild-type Abd-B gene in trans. However, the remaining weak Fab-7 phenotype indicates that UC21-10,1-d is a leaky Abd-B mutation. Imprecise excisions of the insert that result in the deletion of the promoter region of the Abd-B class A transcription unit should eliminate the remaining Fab-7 phenotype in flies heterozygous with the null mutation Abd-B^D16. (The excision of the P element can be detected by the loss of the ry⁺ marker gene carried by the UC21-10,1-d insertion.) We therefore isolated nine ry^- mutations with no remaining Fab-7 phenotype from the progeny (~20,000) of the cross between Fab-7 UC21-10,1-d/TM3 Sb P(ry⁺)2-3 males and Dp(3;1)bxd¹¹¹/Dp(3;1)bxd¹¹¹; ry⁵⁰⁶Abd-B^D16/ry⁵⁰⁶ Abd-B^D16 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-B^D16 chromosome and to establish stocks. Two of them, Abd-B^PSz1 and Abd-B^PSz2, showed significant complementation over iab-7^Sz, i.e., the size of A7 was reduced compared to Abd-B^D16/iab-7^Sz,. Southern analysis detected a deletion of 10.8 kb between map positions +155.6–156.8 and +165.6–166.6 (3.2 kb downstream and 7.6 kb upstream of the site of the original insertion) in Abd-B^PSz1, and a 5.5-kb deletion between map positions +155.6–156.8 and +160.2–161.3 (3.2 kb downstream and 2.3 kb upstream of the site of the original insertion) in Abd-B^PSz2.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (GALLONI et al. 1993 ). Because of the haplo-insufficiency of the Abd-B homeotic gene, disruptions in the normal developmental program of PS12/A7 are evident in animals heterozygous for an iab-7 mutation, such as iab-7^Sz, which deletes the iab-7 region (GALLONI et al. 1993 ). In this case, A7 assumes an identity in between that of the normal A6 and A7. The mixed identity of A7 is manifested as a rudimentary tergite-like structure posterior to A6 with a characteristic shape and size (KARCH et al. 1985 ). This phenotypic transformation is shown in Figure 1B and summarized diagrammatically in Figure 2. A similar effect on A7 development is also observed in animals heterozygous for an Abd-B mutation; however, in this case the disruptions in development are not restricted to A7, and the morphology of the adjacent segments A6 and A5 are altered as well.

View larger version (128K): In this window In a new window Download PPT slide   Figure 1. —Abdominal cuticles of adult males. (a) +/+. In wild type, A7 is represented only by a pair of tracheal openings (arrowhead). (b) iab-7Sz/+. The haplo-insuffiency of the iab-7 cis-regulatory region results in the addition of a rudimentary, seventh tergite. (c) Fab-7/+. Ectopic activation of iab-7 in A6 reduces the sixth tergite to the size of the seventh in iab-7Sz/+. (d) Fab-7/iab-7Sz. Because only a single iab-7 is active in A6 and A7 (that of the Fab-7 chromosome), both sixth and seventh tergites are reduced to the size of the seventh in iab-7Sz/+. (e) Fab-7/Abd-BD16. A7 is similar to that of the previous genotype, but the A6 tergite is enlarged. (f) Fab-7/Df(3R)P9. If, in addition to iab-7, the trans copy of Abd-B is also deleted, both A6 and A7 tergites are again similar to those of the genotype Fab-7/iab-7Sz. (Additionally, due to the partial transformation of A5 into A4, lack of dark pigmentation in patches of A5 is also evident.) (g) Fab-7/McpB116iab-7SzAbd-BD16. The size of the sixth tergite is similar to that of the sixth tergite in Fab-7/Abd-BD16 males e, indicating that the presence or the absence of an inactive copy of iab-7 in trans is indifferent for the suppression of the Fab-7 phenotype. However, A7 is enlarged to the size of A6 in Fab-7/McpB116iab-7SzAbd-BD16 trans-heterozygotes. As shown in h, if Fab-7 is replaced with a wild-type chromosome in this genotype (McpB116iab-7SzAbd-BD16/+), A6 becomes similar to wild type, but A7 remains about the same as in Fab-7/McpB116iab-7SzAbd-BD16, indicating that the Fab-7 deletion only ectopically activates the wild-type iab-7 in PS11, but does not alter its ability to trans-regulate Abd-B.

View larger version (27K): In this window In a new window Download PPT slide   Figure 2. .— Diagrammatic recapitulation of the most relevant results (presented in part in Figure 1) and their interpretation. Continuous line represents the active form of iab-6 and iab-7; shaded rectangle, the inactive (closed) form of iab-7; small black dots, the Fab-7 and the putative Fab-8 boundaries; vertical lines, the endpoints of Fab-7 and iab-7Sz deletions; filled circle, the proximal Abd-B promoter; bold arrow, the Abd-B class A transcription unit; and X, the presence of a point mutation. The regulatory interaction between Abd-B and iab-7 is represented by curved arrows. Dotted arrows and question marks symbolize the possibility of mutual trans-regulation when iab-7 cis-regulatory regions are intact and active on both homologues. The width of black oblongs in column 2 roughly corresponds to the size of the tergites of the different genotypes.

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 (GYURKOVICS et al. 1990 ). Although disruptions in A6 development are also found in Fab-7/+ males, the extent of the transformation is reduced compared to that observed in homozygous mutant animals. Instead of the PS12/A7 identity seen in homozygotes, PS11/A6 assumes a mixed PS11/A6-PS12/A7 identity in animals heterozygous for the Fab-7 mutation. In fact, the mixed identity phenotype of PS11/A6 observed in Fab-7/+ males closely resembles the mixed identity phenotype of PS12/A7 observed in iab-7^-/+ (or Abd-B^-/+ ) male flies (see Figure 1C and Figure B).

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-7^Sz 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-7^Sz 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-B^D16. 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-B^D16 males would be expected to have the same intermediate phenotype in A6 as was observed in Fab-7/iab-7^Sz 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-B^D16 males differs from that in Fab-7/iab-7^Sz males. The A6 tergite in the Fab-7/Abd-B^D16 genotype is enlarged relative to the A7 tergite, and more closely resembles the wild-type A6 segment. This unexpected finding suggests that, unlike iab-7^Sz, the Abd-B^D16 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-B^D16; precisely the same A6 phenotype was observed when other Abd-B point mutations (Abd-B^S1, Abd-B^S4; TIONG et al. 1985 ) were combined with Fab-7 (data not shown).

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-7^Sz 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-7^Sz (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-B^D16, that is, for Fab-7*/Abd-B^D16 males in which the A6 tergite more closely resembled the A7 tergite. [We used Abd-B^D16 as a representative allele in this and subsequent experiments because it is a molecularly characterized mutation that does not code for a detectable protein (BOULET et al. 1991 ).] Out of ~3000 mutagenized chromosomes, we recovered 12 independent Fab-7*/Abd-B^D16 males in which the A6 tergite was as thin or nearly as thin as the A7 tergite. For further analysis, we established stocks of these 12 Fab-7* chromosomes.

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 (LEWIS 1955 , LEWIS 1985 ). The 12 Fab-7* chromosomes were tested for their ability to interact with the Cbx¹Ubx¹ chromosome, and we found that all but one significantly suppressed the wing phenotype induced by the Cbx¹ mutation. Moreover, the strength of the phenotypic effects of these 11 Fab-7* chromosomes closely paralleled that found in combination with Abd-B^D16: mutations that exerted a strong effect on the Fab-7/Abd-B^D16 phenotype also strongly suppressed the wing phenotype (not shown).

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-1^Sz,Fab-7 to TSR-11^Sz,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 LEWIS 1955 for chromosomal aberrations that disrupt transvection in BX-C. The one exception, TSR-12^Sz,Fab-7, proved to be an iab-4 mutation, and it has an inversion breakpoint in BX-C (see Table 1). Although this rearrangement should disrupt pairing in the Abd-B region of BX-C, it would not be expected to affect pairing in the Ubx region of BX-C.

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-B^D16 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 supposition—that the regulatory capacity of iab-7 is limiting—seems 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-B^D16), 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 GALLONI et al. 1993 ). In contrast, such trans-interactions might not be as strong in A7 because cis-interactions between the Abd-B gene and the iab-7 regulatory domain on the same homologue would reduce the ability of the Abd-B promoter to interact in trans. This model could potentially explain the phenotypic difference between A6 and A7 in Fab-7/Abd-B^D16 males (see Figure 2).

If this model is correct, cis-competition in segment A7 should be eliminated by deleting the iab-7 regulatory domain on the Abd-B^D16-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-7^Sz, onto the Abd-B^D16 chromosome. The phenotype of +/iab-7^SzAbd-B^D16 and Fab-7/iab-7^SzAbd-B^D16 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-7^SzAbd-B^D16 males (Figure 1G), and this segment closely resembles the A6 segment found in Fab-7/Abd-B^D16 males (Figure 1E). The similarity between these two mutant combinations would argue that the iab-7 cis-regulatory domain on the Abd-B^D16 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 (GALLONI et al. 1993 ). Second, segment A7 in both +/iab-7^SzAbd-B^D16 (Figure 1H) and Fab-7/iab-7^SzAbd-B^D16 (Figure 1G) males is even more strongly transformed toward an A6 identity than it is in +/Abd-B^D16 or in Fab-7/Abd-B^D16 males. In fact, A6 and A7 are indistinguishable in Fab-7/iab-7^SzAbd-B^D16 males and closely resemble the appearance of A6 in Fab-7/Abd-B^D16 and A7 in +/iab-7^SzAbd-B^D16. It should be emphasized that this transformation in A7 identity cannot be attributed solely to the lack of iab-7 and Abd-B function on the iab-7^SzAbd-B^D16 chromosome. Thus, the transformation of A7 toward A6 in Fab-7/iab-7^SzAbd-B^D16 males is much more extreme than it is when Fab-7 is combined with deficiencies e.g., Df(3R)P9, Figure 1F, or Df(3R)C4, not shown, which completely remove both iab-7 and Abd-B (compare Figure 1G and Figure F). As expected, if the Fab-7 chromosome is replaced by its rearranged version in Fab-7/iab-7^SzAbd-B^D16, i.e., in TSR,Fab-7/iab-7^SzAbd-B^D16 (not shown), the phenotype of A6 and A7 becomes indistinguishable from that of TSR,Fab-7/Abd-B^D16 or Fab-7/Df(3R)P9 (shown in Figure 2 and Figure 1F, respectively).

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-B^D16 and then examined the phenotype of Fab-7/Fab-7Abd-B^D16 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-B^D16 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-7^Sz Abd-B^D16 and Fab-7/Abd-B^D16. 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-B^D16 flies, we see no trace of an A6 to A7 transformation in Fab-7Abd-B^D16/+ flies. Similarly, Abd-B^D16 does not complement iab-7^Sz to any detectable degree in A7 (Figure 6A).

View larger version (68K): In this window In a new window Download PPT slide   Figure 3. —Adult cuticles of (a) Fab-7Abd-BD14/+, (b) TSR-10Sz,Fab-7Abd-BD14/In(3LR)TM3, Sb Ser females. When pairing of the homologues is normal a, A6 is weakly transformed toward A7 as evidenced by the presence of characteristics that are normally found on the wild-type A7, such as hairs pointing toward the middle of the sixth sternite and the absence of trichomes around the sternite (indicated by arrowheads). When pairing is efficiently disrupted (by the rearrangements TSR-10Sz and In(3RLR)TM3), this mild Fab-7 phenotype is no longer detectable b. In addition, the haplo-insufficient phenotype in A7 is clearly stronger (the size of the seventh sternite is enlarged) than in a, indicating that the hyperactivation of the Abd-B gene is also eliminated in the absence of somatic pairing.

View larger version (91K): In this window In a new window Download PPT slide   Figure 4. —Abdominal cuticles of adult males of the genotypes (a) Fab-7/Abd-BD16, (b) Fab-7/Abd-BD14 and (c) TSR-1Sz,Fab-7/Abd-BD14. Suppression of the Fab-7 phenotype by the deletion mutant Abd-BD14 is significantly weaker (but still detectable) than in the combination with the point mutation Abd-BD16 (compare the size of the sixth tergites in a, b and c). Due to the hyperactivation of the wild-type Abd-B gene, the haplo-insufficient phenotype of A7 is suppressed in b (A7 tergite is missing and only a single hair at the tracheal opening, indicated by arrowhead, shows that it is not completely wild type). When pairing is efficiently disrupted c, both hyperactivation and trans-suppression are eliminated, and A6 and A7 tergites are equally thin. The thickness of arrows in the drawings under each of the abdomens indicates the relative strength of cis- and trans-regulation.

View larger version (13K): In this window In a new window Download PPT slide   Figure 5. —Structure of Abd-B deletions used in complementation tests with the deletions iab-7Sz and Df(3R)R59, shown on the molecular map. Continuous bold line indicates the regions covered by the deletions (KARCH et al. 1985 ; GYURKOVICS et al. 1990 ; GALLONI et al. 1993 ; HOPMANN et al. 1995 ); dashed line, uncertainty in the locations of the endpoints; triangle, the position of the insertion UC21-10, l-d (MCCALL et al. 1994 ). Transcriptional unit of class A and part of class B, C and are indicated under the molecular map (ZAVORTNIK and SAKONJU 1989 ).

View larger version (68K): In this window In a new window Download PPT slide   Figure 6. —Abdominal cuticles of adult males of the genotype: (a) Sb Abd-BD16/iab-7Sz, (b) Abd-BD14/iab-7Sz, (c) Abd-BRD18/iab-7Sz and (d) Df(3R)U110/iab-7Sz. The point mutation Abd-BD16 does not complement iab-7Sz to any detectable degree, as A7 appears to be completely transformed into A6. However, the size of the A7 tergite and sternite is progressively reduced in b–d as compared to a, indicating that deletions that remove increasingly larger regions upstream of the Abd-B class A transcription unit complement the iab-7Sz mutation to increasingly higher degrees. In the right column, the diagrammatic interpretation of the phenotype for each genotype is presented. The thickness of curved arrows corresponds to the increasing strength of trans-interaction (cis-interaction is not shown).

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-B^D14 (m^-; KARCH et al. 1985 ), which lacks sequences around the proximal promoter (BOULET et al. 1991 ). As anticipated, we found that Abd-B^D14 weakly complements the iab-7 loss-of-function mutant iab-7^Sz in A7 (Figure 6B). Moreover, unlike in Fab-7Abd-B^D16/+, we were able to detect a weak transformation of A6 into A7 in Fab-7Abd-B^D14/+ adults. This phenotype of weak trans-interaction is best seen in the sixth sternites of females (see Figure 3A).

To rule out the possibility that this weak transformation is due to a "leakiness" of the Abd-B^D14 mutation, we examined the phenotype of Fab-7Abd-B^D14/Df(3R)P9 hemizygotes and Fab-7Abd-B^D14/Fab-7Abd-B^D14 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-B^D14/+ 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-B^D14/iab-7^Sz and Fab-7Abd-B^D14/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-B^D14 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-B^D14/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-B^D14/+ 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-B^D14/TSR,Fab-7 (Figure 4C) and Abd-B^D16/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-B^D14 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-B^D14 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 (MCCALL et al. 1994 ) just upstream of the transcription start site of the Abd-B class A transcript. By itself, this insertional mutation, like the promoter deletion in Abd-B^D14, weakly enhances iab-7 trans-regulation. Thus, Fab-7UC21-10,1-d in trans to a wild-type Abd-B gene transforms A6 toward A7. That this transformation of A6 to A7 is not due simply to the residual Abd-B activity of the UC21-10,1-d allele, but rather arises from trans-activation of the wild-type Abd-B gene, is suggested by the finding that the A6-A7 transformation is strongly reduced when Fab-7UC21-10,1-d is combined with the point mutant Abd-B^D16 (lethality covered by Dp(3;1)bxd¹¹¹).

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-B^PSz1 and Abd-B^PSz2) 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-B^D14. Genomic Southern blotting revealed that Abd-B^PSz1 has a 10.8-kb deletion between map position +155.6–156.8 and +165.6–166.6, and Abd-B^PSz2 carries a smaller deletion of about 5.5-kb from +155.6–156.8 to +160.2–161.3 (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-B^PSz1/+ > Fab-7Abd-B^PSz2/+ > Fab-7Abd-B^D14/+ > Fab-7Abd-B^D16/+. Interestingly, precisely the same relationship is observed for the complementation of the iab-7 deletion, iab-7^Sz, in A7. Thus, the strongest complementation is observed in Fab-7Abd-B^PSz1/iab-7^Sz flies, which is followed by Fab-7Abd-B^PSz2/iab-7^Sz, Fab-7Abd-B^D14/iab-7^Sz, and finally, Fab-7Abd-B^D16/iab-7^Sz. 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-7^Sz in A7. Using this assay, we tested the trans-regulating ability of three previously isolated deficiencies, Df(3R)C4 (KARCH et al. 1985 ), Df(3R)U110 and Abd-B^RD18 (HOPMANN et al. 1995 ) that remove the distal part of BX-C. As expected, Df(3R)C4, which removes iab-7 and part of iab-6 in addition to Abd-B, does not show any complementation. However, both Df(3R)U110 and Abd-B^RD18, which leave iab-7 intact, complement iab-7^Sz to a high degree (Figure 6D and Figure C, respectively). As expected, these two deletions do not suppress the Fab-7 phenotype in trans, and the phenotype of A6 in Fab-7/Df(3R)U110 and Fab-7/Abd-B^RD18 (not shown) is similar to Fab-7/+ (see Figure 1C).

The approximate limits of the complementing deletions are shown in Figure 5. These deletions can be ordered in increasing strength of iab-7^Sz complementation as follows: Abd-B^D14 < Abd-B^PSz2 < Abd-B^RD18 Abd-B^PSz1. 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 (BUSTURIA and BIENZ 1993 ).

We note that unlike Abd-B "point mutations," the Abd-B^PSz2, Abd-B^RD18 or Abd-B^PSz1 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-B^D14, because a weak A7 to A6 transformation (the presence of at least a single bristle in tergite A7; Figure 4B) is observed in Abd-B^D14/+ 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-B^D14 (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-7^Sz. 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 HOPMANN et al. 1995 and HENDRICKSON and SAKONJU 1995 . All of the other Abd-B deletion mutations complement the R59 deficiency in A7 to a degree roughly similar to that observed for iab-7^Sz (data not shown). The lack of this complementation between Df(3R)U110 and Df(3R)R59 does not seem to be due to the fact that these two deficiencies overlap in the region just 3' to the Abd-B gene. Like Df(3R)U110, the deletion in Abd-B^RD18 overlaps Df(3R)R59 in this 3' region, yet Abd-B^RD18 shows the same degree of complementation of Df(3R) R59 as that observed for the Abd-B^PSz1, which does not overlap.

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-B^RD18/iab-7^Sz (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-B^RD18/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-7^Sz, 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-7^Sz is essentially the same as that of Abd-B^RD18/iab-7^Sz, shown in Figure 7A.) Thus, the difference between the phenotype of Df(3R)U110/iab-7^Sz 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.

View larger version (21K): In this window In a new window Download PPT slide   Figure 7. —The position of the iab-7 regulatory region and the Abd-B class A transcription unit (marked with bold line and arrow, respectively) in different pair-wise combinations of deletions. Continuous line represents DNA; parentheses indicate the breakpoints of deletions. (A) nonoverlapping; (B, C) overlapping combinations of deletions. Note that in B the regions marked with bold are relatively close to each other, and in C they are localized at the far ends of the unpaired region.

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 (KAUFMAN et al. 1973 ; MICOL and GARCIA-BELLIDO 1988 ), we tested whether zeste is also required for trans-regulatory interactions involving the Abd-B gene. Surprisingly, we found that mutations in the zeste locus (z^a, z^op6, z¹, z^v77h) had no apparent affect on any of the trans-regulatory interactions in the Abd-B domain described above. HENDRICKSON and SAKONJU 1995 and HOPMANN et al. 1995 have also found that zeste function is not required for the long-range pairing-insensitive trans-interaction that they have uncovered in the Abd-B domain.

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 (CELNIKER et al. 1989 , CELNIKER et al. 1990 ; DELORENZI and BIENZ 1990 ). In wild type, ABD-B protein increases in a stepwise fashion between PS10 and PS14 (CELNIKER et al. 1989 ; SANCHEZ-HERRERO 1991 ; see also Figure 8A). In Fab-7 homozygous embryos, the wild-type pattern of ABD-B protein is observed in all parasegments except for PS11, where Abd-B is expressed in a PS12-like pattern (not shown; see GALLONI et al. 1993 ). As anticipated, no protein can be detected when the Fab-7 embryos are also homozygous for the Abd-B point mutation, Abd-B^D16 (not shown; BOULET et al. 1991 ). In the case of Fab-7Abd-B^D14 homozygotes, protein is observed in the parasegment PS14, but not in PS10–PS13 (Figure 8B). This is expected because the Abd-B^D14 deletion removes the transcription start site of the class A Abd-B transcripts, which are specifically expressed in PS10–PS13. Finally, there is no ABD-B protein in PS10–PS12 of embryos homozygous for the deletion Df(3R)R59, whereas the normal pattern of protein staining is observed in PS13 (where the expression of Abd-B is under the control of the iab-8 cis-regulatory domain) and in PS14 (Figure 8C).

We next examined the Abd-B expression pattern in Fab-7Abd-B^D16/Df(3R)R59 and Fab-7Abd-B^D14/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-B^D14 deletion mutant, but not in the embryos carrying the Abd-B^D16 point mutation. For Fab-7Abd-B^D14/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-B^D16/Df(3R)R59 animals, trans-regulation could be detected in the embryonic CNS. As in Fab-7Abd-B^D14/Df(3R)R59 embryos, we found the same levels of ABD-B protein in the CNS in PS11 and PS12 of Fab-7Abd-B^D16/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-B^D14/Df(3R)R59 and Fab-7Abd-B^D16/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 Mcp^B116iab-7^SzAbd-B^D16/Df(3R)R59 embryos. The Mcp^B116 mutation is a deletion that removes a putative boundary between iab-4 and iab-5, ectopically activating the PS10-specific iab-5 in PS9 (KARCH et al. 1994 ). In animals carrying a wild-type Abd-B gene in cis, the ectopic activation of iab-5 results in the appearance of ABD-B protein in PS9 of the embryonic CNS at a level normally found in PS10 (SANCHEZ-HERRERO 1991). As iab-7 is also deleted on this Mcp^B116 chromosome, Abd-B expression in both PS11 and PS12 can only be driven by the iab-6 regulatory domain. Since the Abd-B gene in cis to Mcp^B116 and iab-7^Sz does not code for a detectable protein, ABD-B protein can only be expressed in these embryos by trans-regulation of the Abd-B gene on the Df(3R)R59 chromosome in PS9–PS12. As can be seen in Figure 8E, we can detect weak ABD-B expression in PS11–PS12, and an even weaker, but unmistakable expression is seen in PS9–PS10. This pattern of protein expression argues that the iab-5 and iab-6 cis-regulatory domains on Mcp^B116iab-7^SzAbd-B^D16 chromosome are capable of trans-regulation much like the iab-7 regulatory domain.

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-B^D14 or Fab-7Abd-B^D16 and chromosomes that have rearrangements that disrupt Abd-B and Ubx trans-regulation in the adult. These rearrangements include TSR-11A, TSR-59A, iab-7^MX2 (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-B^D16/iab-7¹⁶⁴ and Fab-7Abd-B^D14/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-7^R5 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 PS10–PS12 in the CNS. This is the case in embryos homozygous for the In(3R)Fab-7iab-7^R5 inversion; we only see Abd-B staining in PS13–PS14 (not shown). Surprisingly, however, a different result is obtained when In(3R)Fab-7iab-7^R5 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-7^R5 with another inversion, In(3R)Fab-7iab-7^R7, which has its breakpoints in 87D1,2 and 89E3,4 (GYURKOVICS et al. 1990 ). Because these two inversions have similar but not precisely identical breakpoints, they will be paired over much of their length without forming the loop that is typically generated when an inversion chromosome pairs with a structurally wild-type homologue. However, at the proximal inversion breakpoint of one chromosome, there will be a locally unpaired DNA segment that is homologous to an unpaired DNA segment at the distal breakpoint of the other chromosome (see Figure 9). Thus, at the distal end of R5 and the proximal end of R7, there will a large unpaired DNA segment containing the 87C interval. Similarly, at the proximal end of R5 and at the distal end of R7, there will be a short ~10-kb unpaired DNA segment from BX-C that is located just downstream of the Abd-B transcription unit (between map position +141.5–143 and +152) (see Figure 9). If these unpaired DNA segments are able to facilitate long-distance regulatory interactions, Abd-B expression should not be restricted to PS13 and PS14 but instead should also be observed in PS11 and PS12. The results we obtained are consistent with this model. As illustrated in Figure 8G, Abd-B expression can be detected in PS11 and PS12 in the CNS of the R5/R7 embryos, but not in embryos homozygous for either the R5 or the R7 inversion. The expression pattern in the R5/R7 trans-heterozygous embryos is somewhat irregular, suggesting that trans-regulation occurs in some cells but not in others. (We suspect that the Abd-B expression in these embryos may be due to the activity of iab-7 on the R5 chromosome alone. This possibility is suggested by the observation that there is no detectable ABD-B protein in PS10–PS12 of In(3R)Fab-7iab-7^R7/Df(3R)P9 or In(3R)Fab-7iab-7^R7/Df(3R)R59 embryos. This observation would argue that iab-7 on the R7 chromosome is inactivated by the breakpoint in the BX-C.)

View larger version (24K): In this window In a new window Download PPT slide   Figure 8. —Immuno-histochemical staining of ABD-B proteins in the ventral trunk region of the CNS dissected from Drosophila embryos. (a) wild type (+/+). Staining gradually increases from PS10 to PS14. (b) Fab-7Abd-BD14/Fab-7Abd-BD14. Staining is missing from PS10 to PS13. The intensity of staining in PS14 is similar to that in wild type. (c) Df(3R)R59/Df(3R)R59. Staining is missing from PS10 to PS12. (d) Fab-7Abd-BD14/Df(3R)R59. A very low level of protein is detected in PS10 and stronger staining of equal intensity can be seen in PS11 and PS12, indicating trans-regulation of the wild-type Abd-B. (e) McpB116iab-7SzAbD-BD16/Df(3R)R59. Weak staining of equal intensity is detected in both PS9 and PS10, and stronger staining is apparent in both PS11 and PS12. (f) In(3R)Fab-7iab7R5/Df(3R)P9. Weak staining is observed in PS11 and PS12, indicating that, although the paracentric inversion In(3R)Fab-7iab7R5 separates the cis-regulatory region iab-7 from the Abd-B promoter, they can interact via formation of a loop in hemizygous condition. (g) In(3R)Fab-7iab-7R5/In(3R)Fab-7iab-7R7. Weak staining with irregular pattern is detected in both PS11 and PS12, indicating trans-regulation in some cells. (h) In(3LR)iab-7164/Fab-7Abd-BD16. The doubly rearranged chromosome In(3LR)iab-7164 efficiently disrupts pairing of homologous chromosomes, and staining in PS11 and PS12 is significantly decreased as compared to d. Additionally, the pattern of the remaining staining is irregular, indicating that trans-regulation occurs only in some of the cells. Figure 9. —The structure of chromosome pairs carrying different combinations of the inversions In(3R) Fab7iab-7R5, In(3R)Fab-7iab-7R7 and In(3R)Fab-7R5LR7R. Some of these combinations exhibit trans-regulation (marked by +) in the embryonic CNS, and others do not (-). Continuous lines represent chromosomes, short vertical lines the breakpoints of the inversions, and bold arrows the Abd-B class A transcription unit. Thin arrows show the position and the orientation of cis-regulatory regions of Abd-B. Regions within dashed areas are supposed to play a crucial role in transvection. Note that in trans-regulating combinations (+) these regions are unpaired at least at one of the breakpoints.

These findings indicate that unpaired chromosomal DNA segments can facilitate long-distance interactions. One plausible hypothesis is that the two large unpaired 87C chromosomal DNA segments may be able to interact with each other (a form of "homologous pairing"), and it is this interaction that brings the inverted BX-C regulatory regions in close proximity to the Abd-B gene (see Figure 9). This hypothesis is supported by our finding that in ~50% of salivary gland polytene chromosomes, the In(3R)Fab-7iab-7^R5 and In(3R)Fab-7iab-7^R7 homologues form a "ring" or "circle," apparently held together by pairing between the two 87C loops (not shown).

A prediction of this hypothesis is that long-distance regulatory interactions should only be observed when there are unpaired 87C chromosomal DNA segments at both the proximal and the distal breakpoints. To test this prediction, we recombined R5 and R7 to generate a chromosome, R5^LR7^R, which contains the proximal breakpoint of R5 and the distal one of R7. In the R5^LR7^R chromosome, the 87C region is deleted, and a ~10-kb BX-C segment 3' to the Abd-B gene is present at both breakpoints in an inverted orientation. When the R5^LR7^R chromosome is combined with the R5 chromosome, there is only a single, unpaired 87C DNA segment located at the distal R5 breakpoint (see Figure 9). As anticipated, R5^LR7^R/R5 trans-heterozygotes differ from R5/R7 trans-heterozygotes in that they do not form a "circle" in salivary gland polytene chromosomes (data not shown). However, even though this "circle" cannot be detected, the Abd-B expression pattern in PS11 and PS12 of R5^LR7^R/R5 animals is indistinguishable from that observed in R5/R7 animals (data not shown). This is also true for the combination of R5^LR7^R/R7, which has only a single, unpaired 87C DNA segment, in this case at the proximal R7 breakpoint.

Hence, contrary to our expectations, the regulation of Abd-B by a distant iab-7 regulatory domain does not appear to require interactions between unpaired 87C chromosomal DNA segments at the proximal breakpoint of one homologue and the distal breakpoint of the other. On the other hand, there is still a requirement for an unpaired DNA sequence because no long-distance regulation is observed when R5^LR7^R chromosome is homozygous (see Figure 9). We suspect that this unpaired sequence is most likely the short 10-kb BX-C DNA segment immediately downstream of the Abd-B gene. As mentioned above, in the R5/R7 combination there is an unpaired copy of this DNA sequence at the proximal breakpoint of one homologue that could potentially interact with a copy of the same sequence at the distal breakpoint of the other homologue (see Figure 9). In the R5^LR7^R chromosome, there are two copies of this sequence, one at each breakpoint. Consequently, when R5^LR7^R is combined with either R5 or R7 there are three copies of the sequence (see Figure 9). One of these is always unpaired and could potentially interact with the paired copies at the opposite breakpoint. (In this hypothesis, the presence of a single, unpaired 87C region at one end of the otherwise paired combinations of R5^LR7^R/R7 or R5^LR7^R/R5 may only facilitate the looping out of BX-C material and would not play a major role in long-distance regulation.)

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Classical transvection in the Abd-B domain:
Recently, an unusual type of transvection has been demonstrated at the Abd-B domain in the adult stage (HENDRICKSON and SAKONJU 1995 ; HOPMANN et al. 1995 ). This weak trans-interaction between the regulatory regions of the Abd-B domain and the Abd-B gene itself is nearly insensitive to rearrangements that disturb homologous pairing. However, this is not the only type of trans-regulation in this region of BX-C. In the studies reported here, we have detected a more regular type of transvection in the adult cuticle by taking advantage of the dominant gain-of-function mutation Fab-7.

Transvection is usually seen as a partial complementation between a mutation in the cis-regulator and another one in the coding region of the same gene in trans (BENDER et al. 1983 ; ST. JOHNSTON et al. 1990 ; LEISERSON et al. 1994 ). However, in the adult animal we have been unable to see any degree of complementation in such mutant combination, that is, between the cis-regulatory region mutant iab-7^Sz and the Abd-B point mutation Abd-B^D16. We attribute this apparent lack of complementation to the strong haplo-insufficiency of Abd-B. Thus, although trans-regulation may increase the level of ABD-B protein in PS12, this effect is insufficient to produce a detectable change in the phenotype of A7. For this reason we have assayed the consequence of trans-regulation indirectly, by the ability of Abd-B point mutations in trans to suppress the Fab-7 gain-of-function phenotype. However, it must be emphasized that we have used Fab-7 in these experiments only as a convenient tool for detecting iab-7 trans-regulation in A6; precisely the same interaction can be shown in A7 using the combination of a wild type and the double mutant chromosome iab-7^SzAbd-B^D16. The transvection detected in this indirect way in the Abd-B domain closely resembles that found in the Ubx domain (LEWIS 1954 , LEWIS 1955 ; MICOL and GARCIA-BELLIDO 1988 ), and it is disrupted by chromosomal rearrangements that also affect the Cbx phenotype of Cbx¹Ubx¹/++ (LEWIS 1985 ; MATHOG 1990 ). Additionally, both transvecting systems respond to deletions or insertions near the respective promoter regions (MARTINEZ-LABORDA et al. 1992 ). In fact, the only significant difference between transvection at the two loci is that, although in most cases of Ubx transvection the product of the zeste gene appears to play an important role (KAUFMAN et al. 1973 ; MICOL and GARCIA-BELLIDO 1988 ), it does not seem to affect transvection in the Abd-B locus (see also HOPMANN et al. 1995 ).

What is the relationship between this classical Abd-B transvection and the pairing-insensitive, long-range trans-interaction (ITR) described for Abd-B by HENDRICKSON and SAKONJU 1995 and HOPMANN et al. 1995 ? We suspect that phenotypes used to detect trans-interactions in each case require quite different levels of Abd-B expression. ITR is detected as the deposition of black pigment in tergites A6 and A7 ("A5/A6 identity"). This change in pigmentation is likely to require only limited amounts of ABD-B protein, which could be produced by a relatively weak trans-interaction. In contrast, we assay trans-regulation by determining how closely a segment approaches the appearance of the wild-type A7, a segmental phenotype likely to require a considerably higher amount of ABD-B, and consequently, a trans-interaction that is not only stronger but much more dependent on proximity. In addition to this quantitative difference, the two transvecting systems also appear to differ qualitatively. In general, ITR is relatively insensitive to chromosomal rearrangements. In those cases in which the rearrangements do cause a significant impairment of trans-interactions, the resulting phenotype is variegated (and presumably clonal) (HOPMANN et al. 1995 ). In contrast, suppression of the Fab-7 phenotype by Abd-B point mutations responds to interference with pairing in a different way: rearrangements that weakly interfere with pairing of the BX-C region cause a moderate reduction in the size of tergite A6, whereas rearrangements that cause a severe disruption of pairing result in a strong reduction of the A6 tergite but without any apparent "variegation" (Table 1 and not shown). Therefore, it seems likely that the classical transvection reported here involves a mechanism different from those that mediate the long-range trans-interactions described by HENDRICKSON and SAKONJU 1995 and HOPMANN et al. 1995 .

Cis-regulators appear to be tethered to the Abd-B promoter:
We have found that the Abd-B genes in cis and trans compete for interaction with a single iab-7 regulatory domain. This competition depends upon an extensive region at the 5' end of the Abd-B class A transcription unit. Deletion of this region greatly enhances activation of the Abd-B gene in trans by the iab-7 regulatory domain on the deletion-bearing chromosome. The strength of this activation seems to be correlated with the size of the deletion. For example, although the Abd-B^D14 mutation, which deletes just the 5' start site for the class A transcription unit, enhances trans-interactions of the iab-7 in cis, its effect is rather modest compared to deficiencies that remove more extensive regions upstream of the Abd-B class A transcription unit (see Figure 6). Additionally, unlike these larger deletions, Abd-B^D14 suppresses the Fab-7 phenotype in A6 to some extent in trans (compare Figure 4B and Figure C). These findings indicate that Abd-B^D14 is still capable of competing with the wild-type Abd-B gene and suggest that important determinants of competition are located farther upstream. Although we cannot determine the maximum extent of the region that mediates competition from the present data, our deletion analysis suggests that at least 7.6 kb of DNA may be involved.

Why do these 5' deletions enhance trans-activation by the iab-7 regulatory domain (cis to the deletions)? One possibility is that they disrupt pairing between the Abd-B genes on each homologue, allowing the 5' region upstream of the Abd-B promoter on the wild-type homologue to loop out. This looping out would then facilitate trans-regulation. Although we cannot exclude this model, we consider it unlikely. In particular, one expectation of the pairing disruption model is that trans-regulation should be dependent upon the overall size of loop and the relative position of the promoter within the loop. By this hypothesis, Abd-B^RD18 should be more efficient in trans-regulation than Abd-B^PSz-1. First, the Abd-B^RD18 deletion is larger than Abd-B^PSz-1. Second, the promoter is located in the middle of the loop in Abd-B^RD18, and it is at one end of the loop in Abd-B^PSz-1. However, this prediction is not correct: iab-7^Sz is complemented by Abd-B^PSz-1 at least as well (if not better) as Abd-B^RD18.

An alternative hypothesis is that the 5' flanking region of the Abd-B promoter contains "tethering elements" that normally mediate cis-interactions between the Abd-B gene and the iab-7 regulatory domain or the other iab regulatory domains. When these upstream tethering elements are deleted, cis-interactions are weakened or eliminated and trans-interactions with tethering elements upstream of the Abd-B gene on the other homologue become stronger. A similar tethering mechanism has been proposed to anchor the upstream enhancers of the white gene to the white basal promoter (QIAN et al. 1992 ). In white, the tethering region is thought to be no more than ~95 bp in length. By contrast, the tethering region for the Abd-B gene appears to be larger than 7.6 kb. Moreover, because the strength of trans-regulation increases roughly in proportion to the size of the upstream deletion, it would appear that there may be multiple "tethering elements" distributed through this 5' flanking region.

Why does the Abd-B gene have such a large tethering region? We suspect that several factors may be important. First, the regulatory domains that generate the parasegment specific patterns of Abd-B expression are located at a considerable distance from the promoter. Second, the regulation of Abd-B is exceedingly complex, and in each parasegment elements from a different regulatory domain must interact with and control the activity of the Abd-B promoter. Third, because of the arrangement and parasegment specificity of the iab regulatory domains, at least three of the iab regulatory domains (iab-5, iab-6 and iab-7) must contact the promoter across an intervening DNA segment that contains boundary elements (like Fab-7) and one or more Pc-G-silenced regulatory domains. Moreover, these boundary elements are of sufficient strength to prevent the regulatory domains from contacting heterologous promoters (GALLONI et al. l993).

A fourth function of this extensive tethering region may be to ensure cis-preference/cis-autonomy (MARTINEZ-LABORDA et al. 1992 ). Our data show that interactions between an iab-7 domain and an Abd-B gene on the same homologue are, under normal circumstances, strongly preferred to trans-interaction. However, as has been pointed out by MARTINEZ-LABORDA and his coworkers (1992), it is difficult to see a priori how the distant iab regulatory domains are able to distinguish a promoter in cis from the same promoter in trans, particularly when the homologues are the tightly paired. One possibility is that the tethering element array in the 5' flanking region of the Abd-B gene helps ensure cis-preference by making multiple contacts with corresponding elements in the regulatory domains. Once these interactions are established in cis, it would be difficult to rebuild the same set of contacts with the promoter on the other homologue. Even under conditions favorable for trans-regulation, such as when there is only a single active iab-7 regulatory domain (see Figure 10), our results would suggest that there may only be a partial conversion of the cis-complex into a trans-complex. A similar mechanism could account for the apparent cis-autonomy of Ubx (MARTINEZ-LABORDA et al. 1992 ). However, although the presence of an extensive tethering region in the Abd-B 5' flanking region may help explain how cis-preference is maintained, it does not explain why the cis-complex is established in the first place. It is conceivable that the cis-complex is "inherited" from the early stages of embryogenesis, when the BX-C does not appear to be paired (see under next heading). Alternatively, cis-interactions may be established (or reestablished) at a point in the cell cycle, e.g., early G1, that is prior to the time when the homologues become tightly paired.

View larger version (10K): In this window In a new window Download PPT slide   Figure 10. —Schematic model of trans-regulation in the A6 of Fab-7/+ heterozygotes. Continuous line represents DNA; open rectangle, promoter; open circle, enhancer; shaded oval, inactive form of iab-7; black dot, the Fab-7 boundary; vertical line, the end points of the Fab-71 deletion; bold arrow, the Abd-B class A transcription unit; filled and open triangles, protein complexes bound to DNA near the promoter and the enhancer sequences, respectively. An anchoring complex is supposed to be formed between the enhancer and the promoter-proximal region through cooperative interactions among bound protein complexes. We assume a dynamic equilibrium between the more stable cis-complex (formed earlier in time) and the less stable trans-complex. This is supported by the nonclonal appearance of phenotypes resulting from trans-regulation.

The proposed tethering mechanism may also be utilized in selecting the appropriate cis-regulators by the genes in BX-C. For example, iab-6 does not seem to contribute significantly to the expression of Abd-B in PS12 when the iab-7 regulatory domain is intact (CELNIKER et al. 1990 ). However, when iab-7 is deleted, as in iab-7^Sz, the iab-6 domain takes over, and it controls the expression of Abd-B in both PS11 and PS12 (GALLONI et al. 1993 ). On the other hand, the deletion of iab-7 has no apparent effect on the regulation of Abd-B in PS13 by iab-8. These findings suggest that there is a sequential displacement of one iab regulatory domain by the next in progressively more posterior parasegments. That is, in PS11 the Abd-B promoter is complexed with iab-6. In PS12, iab-7 displaces iab-6 from the promoter complex, while in PS13, iab-7 is displaced from the promoter complex by iab-8. This sequential switching could be explained by a difference in the affinity of the iab regulatory domains for the Abd-B tethering region. For example, iab-7 may be able to interact with a larger number of upstream tethering elements and thus form a more stable complex with the Abd-B promoter than iab-6. This model would explain why trans-regulation of Abd-B by iab-7 is not detectably influenced by the presence or absence of iab-6 (and iab-5) in trans. It would also account for the ability of an ectopically activated iab-7 regulatory domain in a Fab-7 chromosome to regulate not only the cis but also the trans Abd-B gene in PS11. In the latter case, it must be able to overcome cis-preference, displacing the iab-6 domain on the trans homologue from the Abd-B promoter.

Our model implies that interacting partners of the tethering elements at the 5' end of the gene should be present in each of the cis-regulatory regions. Although this may be yet another reason for the large size of regulatory regions in the BX-C, we do not have direct evidence for their existence. Likewise, we do not know the protein components/trans-acting genes involved in the formation of the proposed complex. Based on the expected loss-of-function phenotype of these genes, they are likely to belong to the trithorax group. Perhaps with the help of the highly sensitive Fab-7/Abd-B^point phenotype it will be possible to identify those genes that are specifically involved in anchoring cis-regulators to the Abd-B gene.

Transvection is different in different tissues:
We were unable to detect trans-interactions between the iab-7 (or iab-5-iab-6) regulatory region and the Abd-B gene in either the ectoderm or the developing mesoderm during early to mid-embryogenesis. Although we might not be able to detect small amounts of ABD-B protein in the more anterior parasegments where Abd-B expression is low even in wild type, the complete lack of staining in PS12 of extended germ-band embryos suggests that there is essentially no trans-regulation at this stage of development. A plausible explanation is that BX-C is not sufficiently paired for trans-regulation during early embryogenesis. A similar conclusion has been reached by MARTINEZ-LABORDA and coworkers (1992). These authors found that the early ppx function of Ubx, which is required before 10 hr of development, is not restored by interallelic complementation, even under conditions most favorable for trans-regulation (MARTINEZ-LABORDA et al. 1992 ). In this context it is interesting to note that HIRAOKA and coworkers (1993) found that pairing of the chromosome segment harboring the histone genes is already evident after 2–3 hr of embryogenesis. The apparent lack of transvection in BX-C, even at the extended germ-band stage (at about 7 hr of development), suggests that tight pairing of BX-C might occur even later than in the histone gene cluster.

We did, however, find evidence for trans-regulation at slightly later stages of development in the central nerve cord (see also HENDRICKSON and SAKONJU 1995 ). Moreover, it appears that the trans-interactions observed in the embryonic CNS are considerably more tenacious than they are in the adult abdomen (with the caveat that different assays are used to measure trans-regulation in each case). First, trans-regulation in the embryonic CNS can even be detected when the competing Abd-B gene in cis has a wild-type upstream tethering region like that of the point mutant Abd-B^D16. Second, chromosomal rearrangements that would strongly interfere with trans-regulation in the adult have little or no effect in the embryonic CNS.

In fact, in the embryonic nervous system iab regulatory domains are able to interact with the Abd-B gene over quite large distances. This long-distance interaction is best illustrated by the expression of ABD-B protein in PS11 and PS12 in three inversion combinations, In(3R)R5/In(3R)R7, In(3R)R5/In(3R)R5^LR7^R and In(3R)R7/In(3R)R5^LR7^R. In these inversion combinations, the iab regulatory regions on each homologue are separated from the corresponding Abd-B genes by a chromosomal DNA segment extending from 87C-D to 89E. These trans-regulating combinations of inversions identify a relatively short sequence (~10 kb) just downstream of the Abd-B gene which, when locally unpaired, appears to be able to mediate long-distance interaction (Figure 9). Interestingly, these sequences roughly correspond to the region (termed "transvection mediating region" by HOPMANN et al. 1995 ) that was implicated in the weak but extremely tenacious trans-interaction of Abd-B in the adult (HOPMANN et al. 1995 ; HENDRICKSON and SAKONJU 1995 ). However, in contrast to their findings, we have found that this region can support long-distance interaction in the embryonic CNS even if its unpaired copy is contiguous to the regulated gene Abd-B (as in In(3R)R5/In(3R)R5^LR7^R; see Figure 9), and not to the cis-regulatory regions.

What is responsible for mediating the long-distance interactions in the CNS? One plausible hypothesis is that these interactions are facilitated by Polycomb Response Elements (PREs). PREs are short DNA segments that can initiate the assembly of silencing complexes composed of the Polycomb-group (Pc-G) proteins (SIMON et al. 1993 ). The silencing activity of PREs in mini-white transgene assays typically depends upon cooperative interactions between two copies of the PRE (KASSIS et al. 1991 ; HAGSTROM et al. 1997 ). These cooperative interactions are normally observed when the PRE:mini-white transgene is homozygous; however, cooperative interactions can also be detected when the transgenes are inserted into the genome at sites distant from each other (KASSIS 1994 ; HAGSTROM et al. 1997 ; M. MÜLLER, personal communication). These observations raise the possibility that PREs may play some role in mediating the long-distance transvection that we have observed. In this respect, it is interesting to note that a PRE has been found (M. MÜLLER, K. HAGSTROM and P. SCHEDL, unpublished results) in the region we have identified as essential for long-distance interactions in the embryonic CNS. Because this PRE appears to be in the iab-8 regulatory domain (our unpublished data), its silencing activity need not interfere with the activation of Abd-B anterior to PS13 by iab-7 and other, more proximal cis-regulatory regions. The looping out of the DNA segment containing this PRE could enhance its ability to pair with its distant partner, thus bringing the regulatory regions in proximity to the regulated gene. Alternatively, looping out may facilitate the integration of this PRE into a Polycomb compartment (MESSMER et al. 1992 ) in the nucleus, which would provide the required proximity for the regulatory region(s) and the regulated gene.

	ACKNOWLEDGMENTS

We wish to thank ANITA KISS and EDIT GYÁNYI for technical assistance, WELCOME BENDER for the insertional mutation UC21-10,1-d, SUSANNE CELNIKER for the monoclonal Abd-B antibody 1A2E9 and IAN DUNCAN for providing the deletions Abd-B^RD18 and Df(3R)U110. We also thank ISTVÁN ANDÓ for advice on antibody staining and MARTIN MÜLLER for sharing unpublished results. Thanks are due to the members of the Developmental Biology Group of the BRC, especially to IZABELLA BAJUSZ and GABRIELLA TICK, and also to KIRSTEN HAGSTROM and SUSAN SCHWEINSBERG in PAUL SCHEDL's lab for critical reading of the manuscript. This work was supported by the Hungarian National Science Foundation (OTKA grants T 021051 and T 017010), the Bástyai-Holczer Foundation and the Swiss National Science Foundation. We are also indebted to the Human Frontiers Science Program Organization, which helped make this collaborative project possible at its initial stage.

Manuscript received June 12, 1997; Accepted for publication March 2, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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