action in trans is permitted throughout the Drosophila genome

Ji-Long Chen*, Kathryn L. Huisinga*, Michaela M. Viering*, Sharon A. Ou†, C.-ting Wu†, and Pamela K. Geyer*‡

*Department of Biochemistry, University of Iowa, Iowa City, IA 52242; and †Department of Genetics, Harvard Medical School, Boston, MA 02115

Edited by M. M. Green, University of California, Davis, CA, and approved January 22, 2002 (received for review August 23, 2001) Interactions between paired homologous can lead to exists that reduce or eliminate pigmentation in some or all changes in expression. Such trans-regulatory effects exem- cuticle structures. Interestingly, combinations of certain yellow plify transvection and are displayed by many genes in Drosophila, that decrease pigmentation in the same tissue support in which homologous are paired somatically. Trans- complementation in a pairing-sensitive fashion such that pig- vection involving the yellow cuticle pigmentation gene can occur mentation is restored to near wild-type levels (refs. 16, 23, and by at least two mechanisms, one involving the trans-action of 27; S.A.O., J. R. Morris, and C.-t.W., unpublished results). enhancers on a paired promoter and a second involving pairing- Transvection at yellow involves at least two distinct mechanisms mediated bypass of a chromatin . A system was developed (23, 27). In one mode, the wing and body enhancers of one to evaluate whether the action of the yellow enhancers in trans trans-activate the yellow promoter present on the paired homol- could be reconstituted outside of the natural near telomeric loca- ogous . In the second mode, pairing of two alleles tion of the yellow gene. To this end, transgenic flies were gener- alters the effectiveness of an enhancer block conferred by a ated that carried a yellow gene modified by the inclusion of chromatin insulator. These observations indicate that at least strategically placed recognition sites for the Cre and FLP recombi- some mechanisms involved in transvection may be related nases. Independent action of the recombinases produced a pair of directly to those governing enhancer–promoter interactions. derivative alleles, one enhancerless and the other promoterless, at The features of the yellow gene required for transvection are each transgene location. Transvection between the derivatives not understood fully. In this article we address the contribution was assessed by the degree of interallelic complementation. of chromosomal context in this process. The yellow gene resides GENETICS Complementation was observed at all eight sites tested. These Ϸ110 kb from the X chromosome telomere (28). Because studies demonstrate that yellow transvection can occur at multiple telomeres may play a role in chromosome pairing (29–33), the genomic locations and indicate that the Drosophila genome gen- natural yellow location may be important for establishing the erally is permissive to enhancer action in trans. allele proximity necessary for trans-regulatory interactions. To address this issue, we designed a system to test whether the mode ene expression is controlled by regulatory elements that of yellow transvection that depends on enhancer action in trans Gmodulate transcription in appropriate temporal and spatial can occur at new sites within the genome. This system evaluated patterns. In eukaryotes, these control elements often reside in whether the genome was generally permissive to enhancer action large, complex regions, requiring action over long distances to in trans and provided insights into the extent of the yellow region elicit changes in transcription of a target promoter. In some required for these processes. cases, the control elements of paired homologous genes can Materials and Methods interact in trans to alter gene expression. Such trans-regulatory effects illustrate processes known as transvection. Transvection Plasmid Construction. P[yellowCF] was made by cloning a 15.6-kb was described first in Drosophila (1), which has homologous fragment containing the yellow coding region into the P element chromosomes that are paired somatically. Transvection and transformation vector, Carnegie 4 (34). This fragment included Ϸ Ј Ј related processes have been reported in many organisms besides 7.5 kb of 5 - and 2.1 kb of 3 -flanking DNA and the recom- Drosophila (for example, refs. 2–7, and reviewed in refs. 8–11). bination target sites for the bacteriophage P1 Cre recombinase These observations suggest that transvection may be generally (lox P sites) and the yeast FLP recombinase (FRT sites). Direct Ϫ Ϫ possible for eukaryotic control regions and that these processes repeats of lox P sites, located at 2,880 and 900 bp relative to may be important for the normal regulation of some genes. For the transcription start site, flanked the yellow wing and body enhancers, whereas direct repeats of FRT sites, located at example, chromosomal pairing is proposed to play a role in Ϫ ϩ gene silencing associated with imprinting and X chromosome positions 900 and 1,625 bp relative to the transcription start inactivation (4, 12). site, flanked the yellow promoter (Figs. 1 and 2). The lox P and In Drosophila, interactions between homologous chromo- FRT sites were isolated from the p[SFL] plasmid [a gift of Dan somes can have either a positive or negative effect on transcrip- Hartl and Mark Siegal, Harvard University (35)]. Restriction tion. Examples of negative effects include repression of white mapping confirmed the orientation and position of the recom- gene expression by certain alleles of zeste, trans silencing con- bination target sites in the P[yellowCF]. ferred by the insertion of a block of heterochromatin near the Germ-Line Transformation. Germ-line transformation used the yϪ brown gene, and pairing-dependent silencing, as exemplified by Ϫ 1118 the effects of Polycomb response elements (reviewed in refs. ac w host strain that carries a deletion of the natural 8–11, 13, and 14). Examples of positive effects include events at X-linked yellow gene (25). Unless otherwise noted, all genotypes used in this study carried this yϪ allele. Embryos of this strain the Ultrabithorax, Abdominal B, decapentaplegic, yellow, and eyes ␮ ͞ ␮ ͞ absent genes (1, 15–24). were transformed with 0.5 g ml of P[yellowCF] and 0.2 g ml A useful system for studying the mechanisms involved in yellow positive trans-regulatory effects is the gene, which en- This paper was submitted directly (Track II) to the PNAS office. codes a protein responsible for the dark pigmentation of larval ‡To whom reprint requests should be addressed at: Department of Biochemistry, University and adult cuticular structures. Several independent tissue- Ј of Iowa, Bowen Science Building, Iowa City, IA 52242. E-mail: [email protected]. specific enhancers located 5 of the transcription start site and The publication costs of this article were defrayed in part by page charge payment. This within the single intron are required for yellow expression (Fig. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 1 and refs. 25 and 26). A large collection of yellow mutations §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.062447999 PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3723–3728 Downloaded by guest on October 1, 2021 females carrying P[yellowCF] to males that contained a consti- tutively active Cre recombinase transgene (P[wϩ, Cre], kindly supplied by Dan Hartl and Mark Siegel). Male yϪ acϪ w1118͞Y; P[yellowCF]͞ϩ; P[wϩ, Cre]͞ϩ progeny that displayed mutant pigmentation of wing and body tissue were selected. This phenotype indicated that somatic excision of the wing and body enhancers had occurred and suggested that these males con- tained a deletion event within the germ line. The mutant males were mated to y ϪacϪ w1118͞y ϪacϪ w1118; T (2,3)apXa͞CyO, MKRS females, in which CyO and MKRS are balancer chromo- somes for the second and third chromosomes and carry the dominant Cy and Sb markers, respectively. T (2,3)apXa is a translocation between the second and third chromosomes and carries a dominant apterous mutation (apXa). Progeny with mutant wing and body pigmentation were selected, and homozy- gous P[enhϪ] stocks were established. Promoterless derivatives of P[yellowCF], referred to as P[proϪ], were generated by crossing P[yellowCF] females to Fig. 1. Strategy for the generation of structurally distinct yellow alleles from males that carried a heat shock-inducible source of FLP recom- a parental transposon. The P[yellowCF] transposon carried a yellow gene with binase (70FLP, kindly supplied by Dan Hartl and Mark Siegel). target sites for the Cre (open arrowheads) and FLP recombinases (black Embryos aged 15–39 h were subjected to a 1–2-h 37°C heat arrowheads). The P element ends are shown as boxes labeled 5Ј and 3Ј. Cre recombinase catalyzed excision of the 5Ј wing and body enhancers (ovals), shock, and adult male progeny with a variegated cuticle pheno- producing the P[enhϪ] derivative. FLP recombinase catalyzed excision of the type were selected. This phenotype reflected somatic deletion of promoter region (arrow) and intronic (Br, bristle; T, tarsal claw) enhancers, the yellow promoter from the P[yellowCF] transgene and indi- producing the P[proϪ] derivative. Males that carried either the P[enhϪ] or cated that these males were likely to have similar germ-line P[proϪ] allele were crossed to females that carried either the P[enhϪ] or excision events. Variegated males were mated as described above Ϫ P[pro ] allele, and the wing (W) and body (B) cuticle pigmentation of the for the P[enhϪ] derivatives, and homozygous P[proϪ] lines were resulting female progeny was examined to determine if interallelic comple- established. mentation had occurred. Southern analyses were undertaken to ascertain whether the structures of the P[yellowCF] and derivative alleles were as of the ‘‘wings clipped’’ helper plasmid p␲ 25.7 (36, 37). Trans- predicted. Genomic DNA was isolated from adult flies as formants were identified by the dark pigmentation of cuticular described by Ashburner (40), digested with EcoRI, separated on structures (Table 1). Additional lines were made by mobilization a 1% agarose gel, transferred to a nylon membrane, and hybrid- 32 of the P[yellowCF] using a chromosomal source of transposase ized with a P-labeled random-primed 7.7-kb SalI fragment that P[ryϩ⌬2-3] (99B; ref. 38). Only P[yellowCF] lines in which the encompasses the yellow transcription unit (Fig. 2). P element insertion was homozygous-viable were studied fur- ther. Altogether, eight P[yellowCF] homozygous-viable lines Complementation Tests. All crosses were conducted at 25°C and 70% humidity on standard corn meal͞agar medium. Flies car- with a single P[yellowCF] transposon were studied. The cyto- Ϫ Ϫ logical location of each P[yellowCF] transposon was determined rying a P[enh ] insertion were crossed to flies carrying a P[pro ] by in situ hybridization using the entire yellow gene as a probe insertion, and the progeny were scored for wing and body (39). Of the lines analyzed, three were obtained by germ-line pigmentation to determine whether transgenes at ectopic loci transformation (92A, 100F, and 102B) and five from mobiliza- could support interallelic complementation. Wing refers to wing tion (5B, 18C, 47A, 84A, and 90F). blades, and body refers to pigmentation in the abdominal stripes, not the interstripe abdominal cuticle or thoracic cuticle. In Creation of Derivative Alleles. Enhancerless derivatives of P[yel- general, three females were mated with four to five males, and lowCF], referred to as P[enhϪ], were obtained by crossing crosses were transferred every 3 days. Temperature and crowd- ing were monitored carefully, because both affect pigmentation levels. Pigmentation in the wing and body cuticle was scored in Table 1. Pigmentation scores for P[yellowCF] transformants 3- to 4-day-old females by using a five-point pigmentation scale and derivatives (27). A pigmentation score of 1 represents the null or nearly null Cuticle pigmentation score (wing, body) phenotype, and 5 represents the wild-type or near wild-type Insertion state. Pigmentation scores were assigned by comparing progeny Ϫ Ϫ Ϫ ͞ Ϫ site P[yellowCF] P[enh ] P[pro ] P[enh ] P[pro ] to flies obtained from parallel controls. Intermediate scores ϩ 82f29͞ 1#8 5B 5, 5 2 ,3 1,1 4,4 were determined relative to the pigment levels of y y and 2 1#8 18C 5, 5 3, 3ϩ 1, 1 4, 4 y ͞y females, which in this study corresponded to scores of 3,3 47A 5, 5 2, 2ϩ 1, 1 3ϩ,4 and 4,4 in the wing and body, respectively. Pigmentation scores 84A 5, 5 2, 3 1, 1 4ϩ,4ϩ were determined in both the Wu and Geyer laboratories and 90F 5, 5 2ϩ,3 1,1 4,4ϩ represent an analysis of 20–30 female flies from each of at least 92A 5, 5 2, 2 1, 1 4, 4 three independent crosses. Even under these well defined con- 100F 5, 5 3, 3 1, 1 4, 4 ditions, pigmentation scores varied slightly between progeny 102B 5, 5 3, 3 1, 1 4ϩ,4ϩ obtained in different crosses. Scores listed in the tables represent the most common phenotype observed. Complementation be- Pigmentation levels are reported for heterozygous P[yellowCF] (column 2) Ϫ ͞ Ϫ tween derivatives was judged to occur if the pigmentation score or derivative females (columns 3 and 4) and for P[enh ] P[pro ] females Ϫ ͞ Ϫ (column 5). In all cases, flies carried a deletion of the endogenous yellow gene. of the P[enh ] P[pro ] females was at least one point higher Ϫ Pigmentation scores may vary depending on culture conditions. The scores than that obtained for females heterozygous for the P[enh ] listed are those most frequently obtained. allele.

3724 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062447999 Chen et al. Downloaded by guest on October 1, 2021 Fig. 2. Southern analysis of P[yellow CF] and derivative lines. The restriction maps of the parental P[yellowCF] (A), P[enhϪ] (B), and P[proϪ] (C) transposons are GENETICS shown, with the expected sizes of the EcoRI (R) fragments noted above the map. Other enzyme sites shown are HindIII (H) and BamHI (B). W, wing; B, body; Br, bristle; T, tarsal claw. Genomic DNA from two transgenic lines (100F and 84A) was isolated and digested with EcoRI (D). Southern analysis was completed by using a 7.7-kb yellow fragment (marked probe in A). The two lines differ in the size of the flanking band (*), with 100F showing a 12-kb band and 84A showing a 7.5-kb band. The sizes of other bands are shown at the left of each blot.

Results and Discussion The sizes of these shared bands were as predicted, indicating that We assessed whether yellow transvection involving enhancer each line carried an intact P[yellowCF] transposon. The fourth action in trans could occur outside of the natural location of the band was distinctive for each, because it corresponded to DNA Ј yellow gene to address the broad question of how generally sequences from the 3 end of the P[yellowCF] transposon and a permissive the genome is to trans-regulatory inputs. Previous variable amount of flanking genomic DNA (Fig. 2 A and D). The studies at yellow demonstrated that enhancer action in trans presence of a single variable band confirmed that each P[yel- occurred when the yellow promoter in cis was deleted or com- lowCF] line studied carried a single insertion of the transposon. promised (16, 23, 27, 41). By using this information, a method Enhancerless derivatives were obtained by crossing P[yel- was developed to generate chromosomes bearing structurally lowCF] flies with flies carrying a Cre recombinase transgene and distinct yellow alleles at the same ectopic genomic site such that selecting progeny with reduced cuticular pigmentation (Fig. 1). Ϫ one chromosome carried an allele that lacked its promoter, Heterozygous P[enh ] flies had pigmentation scores ranging which presumably would allow its enhancers to activate in trans, from 2 to 3 in the wing and body (Fig. 3 and Table 1). The levels Ϫ whereas the second chromosome carried an allele that lacked its of cuticle pigmentation in P[enh ] flies were compared with enhancers. those of y82f29 flies, which carry a deletion of the wing and body The parental transposon used in our studies was P[yellowCF] enhancers of the yellow gene at its natural location (23). We Ϫ (Fig. 1). This transposon was introduced into flies that carried a found that heterozygous P[enh ]͞ϩ flies had levels of wing and deletion of the endogenous yellow gene, and eight homozygous- body pigmentation that were higher than those of y82f29 flies, ϩ viable transgenic lines were established (Table 1). Six lines had which had scores of 1,1 in wing and body, respectively (23). insertions within the euchromatic portion of the chromosomal These data suggest that although the major wing and body Ϫ arms (5B, 18C, 47A, 84A, 90F, and 92A), one line had an enhancers were deleted from the P[enh ] allele, low levels of insertion close to a telomere (100F), and one line had an yellow expression still occur in these tissues, presumably because insertion on the fourth chromosome (102B), which contains of sequences within the transgene and͞or the surrounding interspersed euchromatic and heterochromatic domains (42). chromatin. Studies in other laboratories have indicated that the Flies from all lines displayed near wild-type levels of cuticular expression of structurally altered yellow genes can be sensitive to Ϫ pigmentation, with scores of 5 in both wing and body (Fig. 3 and the state of nearby sequences (43, 44). Nonetheless, P[enh ] Table 1). flies had greatly reduced wing and body cuticle coloration when Southern analysis was undertaken to confirm the structural compared with P[yellowCF] flies, and this reduction allowed integrity of the eight P[yellowCF] lines. Southern blots of the distinction of interallelic complementation between derivative DNA samples were hybridized with a yellow fragment that alleles. detected only sequences corresponding to the P[yellowCF] trans- Promoterless derivatives were obtained by crossing P[yel- poson, because the endogenous yellow gene was deleted. In each lowCF] flies with flies carrying an FLP recombinase transgene line, four bands of hybridization were observed. Three of the and selecting progeny with reduced cuticular pigmentation (Fig. bands were shared among all lines, because they correspond to 1). Pigmentation of all heterozygous P[proϪ]͞ϩ flies was re- restriction fragments internal to the P element ends (Fig. 2D). duced to 1 in both the wing and body cuticle (Fig. 3 and Table

Chen et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3725 Downloaded by guest on October 1, 2021 Fig. 3. Body and wing phenotypes of P[yellowCF] flies and derivatives. The top two rows show representative flies from two parental P[yellowCF] transgenic lines (100F and 84A), the corresponding P[enhϪ]͞ϩ and P[proϪ]͞ϩ flies and complementing P[enhϪ]͞P[proϪ] flies. The bottom row shows representative flies that have wild-type (Canton S͞yϪ) or mutant (y2͞y2 and y1#8͞y1#8) wing and body pigmentation and a representative fly that displays interallelic complementation (y2͞y1#8).

1). These scores were indistinguishable from those observed for either the P[enhϪ] or the P[proϪ] allele (Fig. 3 and Table 1). In flies carrying a yellow null allele, y1. lines in which the derivative alleles were located on the auto- Southern analyses of the enhancerless and promoterless de- somes, P[enhϪ]͞P[proϪ] males also had dark wing and body rivative lines were carried out. Only changes in the profile of the cuticles, with levels of pigmentation slightly lower than wild-type bands shared among all P[yellowCF] lines was seen. P[enhϪ] (data not shown). derivatives had a 5.5-kb band of hybridization that replaced the Southern analyses of DNA isolated from P[enhϪ]͞P[proϪ] 4.8- and 3.3-kb bands because of the loss of an EcoRI site in flies confirmed that these darkly pigmented flies carried both the Cre-induced deletion (Fig. 2B). P[proϪ] derivatives retained derivative alleles, because the observed pattern of hybridization the 4.8-kb band of hybridization but lost the 3.3-kb band, be- was a composite of that seen for P[enhϪ] and P[proϪ] flies (Fig. cause the latter fragment resided entirely between the FRT sites 2D). Importantly, this pattern did not include the 3.3-kb band, (Fig. 2C). These Southern analyses confirmed that the structures which is diagnostic of the presence of the parental P[yellowCF] of the P[proϪ] and P[enhϪ] derivatives were as predicted. We transposon, indicating that the observed increase in pigmenta- conclude that the action of the recombinases did not alter the tion resulted from complementation, not contamination. From genomic location of any derivative allele, because the size of the these data we conclude that the trans action of the yellow wing unique flanking bands remained unchanged (Fig. 2D). and body enhancers on a paired promoter is possible at ectopic Flies carrying a P[enhϪ] allele at one ectopic site were crossed locations. with flies carrying the corresponding P[proϪ] allele at the same For reference, the pigmentation levels in P[enhϪ]͞P[proϪ] site, and the level of wing and body pigmentation in the female females were compared with females carrying complementing progeny was assessed to determine whether interallelic comple- yellow alleles at the natural location. These studies used the y2, mentation could occur (Fig. 1). At all eight genomic sites, y1#8 and y1 alleles. The y2 allele is a tissue-specific mutation P[enhϪ]͞P[proϪ] females had levels of wing and body pigmen- caused by the insertion of a gypsy retrotransposon between the tation that were darker than those of females heterozygous for yellow promoter and the wing and body enhancers. Because

3726 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.062447999 Chen et al. Downloaded by guest on October 1, 2021 Table 2. Complementation scores of P[yellowCF] derivatives inserted at the same or different genomic locations Cuticle pigmentation score (wing, body)

Genotype yacw(1B) P[proϪ] 18C P[proϪ] 47A P[proϪ] 84A P[proϪ] 90F P[proϪ] 92A P[proϪ] 100F P[proϪ] 102B

y2 (1B) 1, 1ϩ 1, 1ϩ 1, 1ϩ ND 1, 1ϩ 1, 1ϩ 1, 1ϩ ND P[enhϪ] 18C 3, 3ϩ 4, 4 3, 3ϩ 3, 3 3, 3ϩ 3, 3 2, 3 3, 3 P[enhϪ] 47A 2, 2ϩ 2ϩ,2 3؉,4 2ϩ,2 2ϩ,2ϩ 2, 2 2, 2ϩ 2, 2 P[enhϪ] 84A 2, 3 2ϩ,2ϩ 2ϩ,2ϩ 4؉,4؉ 2ϩ,3 2ϩ,2ϩ 2ϩ,3 2ϩ,2ϩ P[enhϪ] 90F 2ϩ,3 2ϩ,3 2ϩ,3 2ϩ,2ϩ 4, 4؉ 2, 2ϩ ND 2ϩ,3 P[enhϪ] 92A 2, 2 2, 2 2ϩ,2ϩ 2, 2 2, 2 4, 4 2, 2 2, 2 P[enhϪ] 100F 3, 3 3, 3 3, 3ϩ ND 3, 3 3, 3 4, 4 3, 3 P[enhϪ] 102B 3, 3 ND 3, 3ϩ 3, 3 3, 3ϩ 3, 3 3, 3ϩ 4؉,4؉

In these experiments, pigmentation scores are reported for crosses between P[proϪ] males and P[enhϪ] females. Complementing scores are shown in bold. Crosses carried out in the opposite direction gave the same results. ND, not determined.

gypsy carries a chromatin insulator that blocks enhancer- y2͞yϪ; P[proϪ]͞ϩ flies displayed noncomplementing levels of promoter communication, y2 flies have wing and body pigmen- wing and body pigmentation (Table 2), demonstrating that the tation scores of 1,1ϩ, respectively (45). The y1#8 allele carries a ectopic P[proϪ] derivatives failed to support transvection with y2. 780-bp deletion of the promoter region and is a null allele. Flies Next we determined whether a P[enhϪ] allele and a P[proϪ] carrying this allele have scores of 1 in the wing and body (46). allele from different ectopic sites supported transvection. In The y1 allele carries a mutation in the translation start codon, is these studies, P[proϪ] males corresponding to seven ectopic sites a null allele, and also gives scores of 1 in both the wing and body were mated to P[enhϪ] females from these locations. Once (16). Flies carrying both the y2 and y1#8 alleles show comple- again, complementation was not observed (Table 2). These mentation, with near wild-type levels of wing and body pigmen- data indicate the importance of allele proximity for yellow tation (scores of 4,4; Fig. 3). In contrast, flies carrying the y2 and transvection. GENETICS y1 alleles show no complementation, displaying low levels of wing There are many possible reasons why transvection between and body pigmentation (scores of 1ϩ,1ϩ). The level of pigmen- nonhomologous sites was not observed. First, yellow regulatory tation observed in P[enhϪ]͞P[proϪ] flies was similar to that sequences may have difficulty locating each other. In previous observed in y2͞y1#8 flies (Fig. 3). circumstances where interactions occurred between nonho- Transvection effects depend on apposition of the interacting mologous sites, alterations of gene expression occurred most genes. In Drosophila, this proximity is most commonly brought frequently when the interacting pair was located on the same about by chromosomal pairing. A variety of genetic tests chromosome arm (51, 52). In our study, even though three of indicate that loci showing transvection effects differ in their the tested nonhomologous sites met this criterion (90F, 92A, sensitivity to chromosomal rearrangements that are believed and 100F), transvection was not seen. Second, if trans- to disrupt local pairing (1, 15, 18–21, 47–50). Whereas trans- activation of the yellow promoter by the wing and body vection at some loci can be disrupted by rearrangement break enhancers requires prolonged and͞or intimate interactions points up to half a chromosome arm away (1, 15, 18), trans- between these regulatory elements, then it may be that non- vection at other loci can occur even in the presence of break homologous sites cannot establish the required conditions. points within the interacting regions (19–21). In these cases, it Third, the complementation we observed at homologous sites has been proposed that an extensive region of the interacting may have depended partially or fully on pairing forces pro- genes is involved in the capture of regulatory elements (19– vided by the flanking chromosomal regions. If this is the case, 21). Furthermore, some homologous sequences, on the order then the lack of sufficiently strong pairing functions within the of a few kilobases or less, inserted at different chromosomal yellow transgenes and͞or the tenacious pairing of the flanking locations still retain the ability to alter gene expression, regions may have precluded productive pairing between non- presumably through a pairing-mediated mechanism (reviewed homologous sites and prevented transvection. Fourth, our ref. 14). Particularly relevant to our studies is the observation pigmentation assay may not be sensitive enough to detect low that two transgenes located in different chromosomal posi- level complementation between nonhomologous sites. Finally, tions, one carrying the miniwhite gene and the other carrying it is possible that the chromosomal organization within the the white eye enhancer, supported gene activation (51). Al- nucleus may impose constraints on the extent of trans- though this activation was observed in only a fraction of the regulatory interactions exerted between enhancerless and chromosomal sites tested, it indicates that enhancers can promoterless pairs at nonhomologous sites (53, 54). activate promoters even when located in nonhomologous In summary, a method was introduced to address whether genomic sites. Taken together, these data suggest that homol- the Drosophila genome is generally permissive to positive ogous sequences can have a surprising ability to find each other trans-regulatory inputs. Recently, a similar approach ad- and pair in the context of a complex genome. dressed whether homologue pairing that influences gene ex- To examine the capacity of yellow sequences to find each other, pression could occur in transgenic tobacco (7). In that study, we asked whether yellow enhancers located at one chromosomal it was found that in certain circumstances, pairing of an intact site could activate a yellow promoter located elsewhere. First we gene with a homologue lacking its enhancer can increase gene assessed whether the y2 allele at its natural location comple- expression, possibly caused by enhancer action in trans. In our mented promoterless alleles at five ectopic chromosomal loca- studies, we found that yellow transvection between enhancer- tions (Table 2). Importantly, each allele used in these tests less and promoterless alleles was reconstituted at eight of eight supports transvection when it is able to pair with another yellow ectopic sites. These interactions are believed to involve pairing, allele at a homologous chromosomal site (Fig. 3 and Table 1), because interallelic complementation was not observed implying that any lack of complementation with an allele at a between yellow alleles at nonhomologous sites. These data nonhomologous site cannot be attributed to an intrinsic inability indicate that the natural location of the yellow gene is not of these transgenes to support transvection. In all cases, the essential for trans-regulatory interactions and imply that en-

Chen et al. PNAS ͉ March 19, 2002 ͉ vol. 99 ͉ no. 6 ͉ 3727 Downloaded by guest on October 1, 2021 hancer action in trans may be permitted throughout the We thank Mark Siegel and Dan Hartl for P[wϩ, Cre] and 70FLP fly genome. Finally, this paper demonstrates that the Cre-FLP stocks and the p[SFL] plasmid. We thank Jim Morris, Timothy Parnell, system can be used to identify critical sequences that control and Robin Roseman for useful discussions, technical assistance, and the trans action of enhancers in vivo, leading to a better critical reading of the manuscript. This work was supported by National understanding of the mechanisms governing enhancer– Institutes of Health Grants GM42539 (to P.K.G.) and GM61936 (to promoter communication. C.-t.W. and P.K.G.).

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