Replacement of Fab-7 by the gypsy or scs Insulator Disrupts Long-Distance Regulatory Interactions in the Abd-B Gene of the Bithorax Complex

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1 Molecular Cell, Vol. 8, , November, 2001, Copyright 2001 by Cell Press Replacement of Fab-7 by the gypsy or scs Insulator Disrupts Long-Distance Regulatory Interactions in the Abd-B Gene of the Bithorax Complex Ilham Hogga, 1 Jozsef Mihaly, 1,2 Stéphane Barges, 1 and François Karch 1,3 1 Department of Zoology and Animal Biology University of Geneva 1211 Geneva 4 Switzerland 2 Institute of Genetics Biological Research Center 6701 Szeged Hungary Summary Chromatin domain boundaries, like scs or gypsy insulators in Drosophila, have been identified in trans- gene assays through their enhancer-blocking activity. Boundary elements in the bithorax complex (BX-C), such as Fab-7 and Fab-8, have been identified genetically and been shown to have insulator activity in transgene assays. However, it is not clear whether boundary elements identified in transgene assays will function appropriately in chromosomal contexts such as BX-C. Using gene conversion, we have substituted the scs or gypsy insulators for Fab-7. We find that both scs and gypsy are very potent insulators in the ectoderm, but surprisingly, the insulating activity of gypsy (but not scs) is lost in the CNS. Our results reveal that the Fab-7 boundary must have special properties that scs and gypsy lack, which allow it to function appropriately in BX-C regulation. Introduction enhancer/silencer and the promoter, but should be neutral when placed distal to the regulatory element (Kellum and Schedl, 1992). Using assays of this type, elements that can function as boundaries or insulators have been identified in species as diverse as yeast and mouse (for a listing, see Sun and Elgin, 1999). In Drosophila, two boundary elements, scs/scs and the gypsy insulator, have been studied extensively using the position effect and enhancerblocking assays. The scs/scs boundaries flank the two divergently transcribed 87A7 hsp70 genes, and are located at the border of the puff that forms at the heat shock locus upon heat induction (Udvardy et al., 1985; Kellum and Schedl, 1991, 1992; Vazquez and Schedl, 1994; Zhao et al., 1995). The gypsy insulator is a small, 400 bp sequence near the 5 LTR of the gypsy retrotransposon, which contains reiterated binding sites for the zinc finger Su(Hw) protein (Parkhurst et al., 1988). Many of the classical spontaneous mutations in the fly turn out to be due to the insertion of the gypsy transpo- son (Modolell et al., 1983). When the transposon is lo- cated in between distant regulatory regions and the pro- moter, the gypsy insulator disrupts gene function by preventing regulatory interactions (Peifer and Bender, 1986; Jack et al., 1991; Geyer and Corces, 1992). The insulator activity of the gypsy Su(Hw) element has sub- sequently been confirmed in both the position effect and enhancer-blocking assays (Geyer and Corces, 1992; Roseman et al., 1993). While scs/scs and the gypsy insulator have properties consistent with those expected for domain boundaries, there is no direct evidence that these elements actually function to delimit chromatin/regulatory domains in vivo. Perhaps the best evidence that boundary elements delimit higher order chromatin/regulatory domains comes from studies on the Drosophila bithorax complex (BX-C). The three homeotic genes in the bithorax complex, Ultrabithorax (Ubx), abdominal-a (abd-a), and Abdominal-B (Abd-B) are responsible for specifying segment identity in the posterior two thirds of the Drosophila embryo, from parasegments (PS) 5 14 (Lewis, 1978; Sanchez- Herrero et al., 1985). These three genes are regulated by an elaborate cis-regulatory region that spans a DNA segment of over 300 kb. This cis-regulatory region is subdivided into nine functionally autonomous domains, abx/bx, bxd/pbx, and iab-2 iab-8 (Lewis, 1978; for re- views, see Peifer et al., 1987; Duncan, 1987). Each domain specifies the identity of a specific parasegment by activating one of the BX-C homeotic genes in a pattern appropriate for that parasegment. For example, as illus- trated in Figure 1, Abd-B expression in PS10 13 is regu- lated by the four cis-regulatory domains, iab-5 iab-8, respectively, that span a 60 kb DNA segment located downstream of the Abd-B transcription unit (Celniker et Eukaryotic chromosomes are thought to be organized into a series of discrete higher order chromatin domains. This organization is important not only in the compaction of the chromosome fiber, but also for the utilization of genetic information. Implicit in the domain model are elements, the so-called boundaries, that define the limits of each domain and insulate the domain from the regulatory influences of adjacent domains (for reviews, see Gerasimova and Corces, 1996; Geyer, 1997; Sun and Elgin, 1999; Udvardy, 1999; Bell et al., 2001). Two assays have been used to test candidate elements for domain boundary function. The first tests whether candidate boundaries are capable of insulating a reporter con- struct from chromosomal position effects. Since bound- ary elements are thought to delimit domains of independent gene activity, reporter genes flanked on both sides by boundaries should be protected against both positive and negative chromosomal position effects (Kellum and Schedl, 1991). The second assay tests whether candidate boundaries can block regulatory interactions be- al., 1990; Sanchez-Herrero, 1991). tween either an enhancer or a silencer and a nearby In order to properly specify segmental identity, the promoter. In this assay, the boundary should prevent four Abd-B cis-regulatory domains must be able to funcregulatory interactions when interposed between the tion autonomously. Functional autonomy is conferred by boundary elements located in between each of the 3 Correspondence: francois.karch@zoo.unige.ch iab cis-regulatory domains. The best characterized of

2 Molecular Cell 1146 Figure 1. Molecular Map of the Abdominal Region of the BX-C (A) The genomic DNA is marked off in kilobases. The approximate extent of the iab-2 iab-8 cis-regulatory domains are shown as blocks of different colors. The arrows indicate the target promoter (abd-a or Abd-B) for each cis-regulatory domain and the parasegment/segment in which the interaction occurs. The positions of the Fab-7 and Fab-8 boundaries are indicated. (B) Magnification of the Fab-7 boundary region drawn at the scale indicated (see Experimental Procedures). these boundaries are Fab-7 and Fab-8. Fab-7 separates quite similar to other boundary elements such as scs the iab-6 and iab-7 cis-regulatory domains, while Fab-8 and gypsy. When interposed between an enhancer (or separates iab-7 and iab-8 (see Figure 1; Gyurkovics et silencer) and a reporter gene, both Fab-7 and Fab-8 al., 1990; Galloni et al., 1993; Mihaly et al., 1997; Zhou can block regulatory interactions (Hagstrom et al., 1996; et al., 1999; Barges et al., 2000). When one of the bound- Zhou et al., 1996, 1999; Barges et al., 2000). Like scs and aries is deleted, the cis-regulatory domains fuse into a gypsy, this blocking activity shows no stage or tissue new functional unit, and are no longer able to gener- specificity and is observed in embryos and adults. There ate the appropriate parasegment-specific patterns of is also evidence that Fab-7 and Fab-8 even have en- Abd-B expression. For example, deletion of the Fab-7 hancer/silencer-blocking activity in the context of BX-C. boundary fuses the iab-6 and iab-7 cis-regulatory domains. This comes from two reporter genes inserted into the This fusion disrupts Abd-B regulation in PS11. iab-7 cis-regulatory domain, one close to the Fab-7 Usually, it results in the inappropriate activation of iab-7 boundary (the bluetail transposon; Galloni et al., 1993), in PS11 by positive elements in the iab-6 cis-regulatory and the other close to the Fab-8 boundary (fs(3)5649; domain. As a consequence, Abd-B is regulated in a Barges et al., 2000). These reporter genes are subject PS12-like pattern, transforming cell identity from PS11 to the enhancers/silencers in the iab-7 cis-regulatory to PS12. Occasionally, negative elements in iab-7 manage domain, but are insulated from regulatory elements in to silence the fused cis-regulatory domain alto- either iab-6 or iab-8. gether. In this case, Abd-B is regulated by the active In all of these experiments, the only difference that iab-5 cis-regulatory domain, transforming cell identity can be discerned between Fab-7/Fab-8 and scs/gypsy from PS11 to PS10. is that the insulating activity of the two BX-C boundaries Although the normal function of BX-C boundaries like in transgene-based enhancer/silencer-blocking assays Fab-7 and Fab-8 is to prevent adventitious interactions is not quite as strong. However, it is not at all clear that between adjacent cis-regulatory domains and not, for this difference is significant, since other fly boundaries example, to block enhancer-promoter interactions, the like scs and fa swb are also weaker in these same assays available evidence suggests that they have properties than scs or gypsy (Vazquez and Schedl, 1994, 2000).

3 Boundary Swapping in the BX-C 1147 interactions between the iab-5/iab-6 cis-regulatory domains and Abd-B. While previous studies on gypsy- induced mutations in both cut (Dorsett, 1993) and BX-C (Peifer and Bender, 1986) indicate that the gypsy insulator can block regulatory interactions over such a large distance, this is a novel finding for scs. It should be noted here that the insulating activity of gypsy and scs min observed in the Abd-B expression assay and in the assays described below is orientation independent (data not shown). The homeotic transformations seen in adults when Fab-7 is replaced by the gypsy or scs min insulator (Figure 3) agree with the effects on Abd-B expression observed in the embryonic ectoderm. In order to understand these homeotic transformations, it is best to describe first the phenotypic consequences of a rearrangement that breaks between iab-6 and its target gene Abd-B (such as iab-6 11 ; Celniker et al., 1990; Figure 3). In embryos, such chromosomal breaks prevent iab-5 and iab-6 from contacting the Abd-B promoter and as a consequence, Abd-B is not expressed in either PS10 or PS11. In adults, this would be expected to result in a posterior to anterior transformation of both A5/PS10 and A6/PS11, into A4/ PS9. A4/PS9 identity is normally specified by the action of the iab-4 cis-regulatory domain on the abd-a gene (Figure 1). In the absence of Abd-B, only abd-a would be expected to be active in PS10 and PS11, and its expression should be directed by iab-4. However, A5/ PS10 appears normal while A6/PS11 assumes an A5/ PS10 identity (Figure 3). To explain this unexpected re- sult, it has been proposed that when Abd-B is not pres- ent in cis, iab-5 is targeted instead to the abd-a gene, which it activates in a pattern appropriate for specifying a A5/P10-like identity (Celniker et al., 1990; Hendrickson and Sakonju, 1995; I. Duncan, personal communication; H. Gyurkovics, personal communication). Precisely the same phenomenon occurs when Fab-7 is replaced by the gypsy or scs min insulator. Despite the fact that there is little or no Abd-B expression in either A5/PS10 or A6/PS11, A5/PS10 identity is normal and A6/PS11 is transformed into A5/PS10. In this view, both insulators act like a rearrangement break, preventing interactions with Abd-B and instead directing iab-5 to contact and regulate abd-a. There are experimental precedents for insulators redirecting enhancers to alter- native or less-preferred promoters. Ohtsuki et al. (1998) have shown that when enhancers such as IAB5 (from the iab-5 cis-regulatory domain) or AE1 (from the ftz cis- regulatory region) are simultaneously challenged with a TATA-containing promoter and a TATA-less promoter, they preferentially interact with the TATA-containing promoter. However, if the gypsy insulator prevents us- age of the TATA-containing promoter, the IAB5 or AE1 enhancers are able to efficiently activate the available TATA-less promoter. In the context of BX-C, the IAB5 enhancer is closer to the abd-a promoter than to Abd-B. Moreover, Abd-B is driven by a TATA-less promoter. Although the nature of the abd-a promoter remains un- known (TATA-less or TATA-containing), these observa- tions suggest that within BX-C, special elements may target IAB5 on the Abd-B promoter. The insulating activity of the gypsy element in other contexts has been shown to depend on two genes, su(hw) (Parkhurst et al., 1988), which encodes a protein These apparent similarities raise the question of whether scs or gypsy can substitute for Fab-7 in the context of BX-C and generate a functionally independent cisregulatory domain. Making this question especially intri- guing is a paradox posed by the organization of the Abd-B cis-regulatory domains and the Abd-B transcrip- tion unit: the more distal iab-5 and iab-6 cis-regulatory domains must somehow overcome the intervening Fab-7 and Fab-8 boundaries in order to contact the Abd-B promoter and activate the appropriate expression patterns in PS10 and PS11. Thus, a second question is whether the distal cis-regulatory domains can overcome the scs and gypsy boundaries. To address these two questions, we used the P element-mediated gene conversion technique of Gloor et al. (1991). We find that both insulators can revert the dominant gain- of-function Fab-7 phenotype, indicating that they can prevent adventitious interactions between iab-6 and iab-7 and establish a functionally autonomous cis-regu- latory domain. However, the enhancer-blocking activity of the gypsy and scs insulators has a deleterious effect on the activity of more distal cis-regulatory domains (relative to the insertion point within the Fab-7 region) and prevent them from interacting with the Abd-B pro- moter. Results and Discussion Figure 1B shows a magnification of the Fab-7 region and the extent of the Fab-7 2 deletion that was used as the boundary deletion in our replacement experiments. The DNA segment deleted in Fab-7 2 was replaced by either the gypsy or scs min (a minimal scs fragment; see Experimental Procedures) boundary. The boundary se- quences were inserted in either orientation at a site 140 nucleotides upstream from the Fab-7 2 deletion. To characterize the Fab-7 boundary replacements, we ex- amined the pattern of Abd-B expression in the epidermis and CNS of embryos and the phenotype of the adult male abdominal cuticle. The typical wild-type graded Abd-B expression pat- tern in PS10 PS13, with the highest level in the more posterior segments, is shown in Figure 2A. In Fab-7 deletion embryos, Abd-B expression in PS10 is normal; however, in PS11, it is generally PS12-like due to the ectopic activation of the iab-7 cis-regulatory domain by iab-6 (Galloni et al., 1993). The Abd-B expression pattern in embryos carrying the gypsy insulator is quite different from that seen in wild-type or Fab-7 mutants; there is no detectable Abd-B expression in either PS10 or PS11, while the pattern of Abd-B expression in PS12 and PS13 is normal. Two conclusions can be drawn. First, the gypsy element is able to block adventitious interactions between the iab-6 and iab-7 cis-regulatory domains, and in this respect, can apparently substitute for Fab-7. On the other hand, it also prevents the iab-5 and iab-6 cis-regulatory domains from regulating Abd-B expression in PS10 and PS11. As a consequence, Abd-B is completely shut down in these parasegments. Essentially the same result was obtained for the scs min replacements. However, in contrast to gypsy, a low level of residual Abd-B expression can be detected in PS10 and PS11, indicating that scs min does not fully block

4 Molecular Cell 1148 Figure 2. Abd-B Expression Pattern in Wild-Type, gypsy, and scs Homozygous Embryos Abd-B was detected with a mouse monoclonal antibody (Celniker et al., 1990) followed by staining with a peroxidase-coupled secondary antibody. (A) Embryos were opened along the dorsal midline, flattened, and mounted on a microscope slide. Parasegments are indicated. At about 10 hr of development, Abd-B is expressed at its maximum level in the epidermis and appears in some neuroblasts along the midline. In wild-type, the typical Abd-B expression pattern is characterized by an anterior to posterior gradient in the number of positive nuclei per parasegment as well as by the intensity in each nucleus. In homozygous gypsy, there is no detectable signal in PS10 and PS11, indicating that both iab-5 and iab-6 are unable to interact with the Abd-B promoter. In homozygous scs min embryos, a weak signal is detected in PS10 and PS11. (B) Central nervous systems (CNS) that were dissected from 12-hr-old embryos are shown. In scs min embryos, an impediment between iab- 5/iab-6 and Abd-B is visible by the weaker signal in PS10 and PS11. In homozygous gypsy, Abd-B appears in the CNS at levels that are almost as intense as in wild-type, contrasting with the complete absence of detectable staining at an earlier stage in the epidermis and CNS of PS10 and PS11 (A). The level and pattern in PS11 resembles that seen in PS12, indicating that iab-7 is ectopically activated in PS11 (the Fab-7 2 phenotype). that binds to the gypsy element, and mod(mdg4) (Gera- interactions with Su(Hw) (Gause et al., 2001; Ghosh et simova et al., 1995), which encodes a protein that interacts al., 2001), the absence of effect on the insulating activity with Su(Hw). We find that in a su(hw) mutant back- demonstrates that in the context of the Fab-7 region, ground, the gypsy element is no longer able to function Su(Hw) can insulate without the aid of the Mod(mdg4) as an insulator; su(hw) mutant flies heterozygous for protein. the BX-C gypsy element have dominant abdominal While the gypsy and scs min insulators behaved similarly transformations essentially identical to those seen in in the embryonic epidermis and produced a similar homeotic flies heterozygous for a Fab-7 boundary deletion (Figure phenotype in adults, a surprise came when we 4). In contrast, mutations in mod(mdg4) had no detect- examined Abd-B expression in the CNS. As shown in able effect on the insulating activity of the gypsy element, Figure 2B, the gypsy insulator does not block Abd-B and the abdominal transformations are identical expression in the CNS in either PS10 or PS11. Moreover, to those caused by the gypsy element in a wild-type the Abd-B expression pattern in PS11 resembles that background. As the mod(mdg4) allele used (u1) encodes normally seen in PS12 that is, the expression pattern a truncated protein lacking the domain necessary for is like that of a mutant that lacks the Fab-7 boundary.

5 Boundary Swapping in the BX-C 1149 Figure 3. Homeotic Transformations Associated with the Swapping of Fab-7 by the gypsy or scs Insulator Male abdomens were cut along the dorsal midline and flattened on a slide. Only half cuticles are shown in which A4 A6 are well visible. The dorsal surface of each abdominal segment has a rectangular plate of hard cuticle called the tergite (only half of the tergites are visible on the right of each panel, as well as the genitalia at the bottom). Note that in wild-type, the fifth and sixth tergites are pigmented (numbered). The ventral surface of abdominal segments is composed of soft cuticle called the pleura. On the midline of the pleura of A2 A6, there are small plates of harder cuticle called sternites. In wild-type, the sixth sternite (framed by small arrows in all panels) can be easily distinguished from the more anterior sternites by its different shape and by the absence of bristles. In males homozygous for the chromosome carrying the gypsy insulator, the sixth sternite is covered by bristles and has the shape of a fifth sternite, indicating a transformation of A6 into A5. The normal pigmentation of the fifth tergite indicates that A5 has normal identity (see text). In homozygous males in which scs min replaces Fab-7, the phenotypic effects are the same as in the case of gypsy swapping. In iab-6 11 homozygous males, a rearrangement break interrupts chromosomal continuity between iab-6 and iab-7. Although iab-5 is unable to regulate Abd-B, A5 identity appears normal (see text). Thus, in the CNS, the gyspy element can not block com- the cis-regulatory domains and the three homeotic munication between the iab cis-regulatory domains and genes. Evidence for such mechanisms already exists. Abd-B promoter, nor is it able to ensure the functional Studies on transvection in the Abd-B domain indicate autonomy of the iab-7 and iab-6 cis-regulatory domains. that there is a large tethering region upstream of the As the gypsy insulator has been shown to function in the Abd-B promoter that mediates communication with the CNS in other chromosomal contexts (Jack and DeLotto, iab cis-regulatory domains (Sipos et al., 1998). One func- 1995; Hagstrom et al., 1996), this observation suggests tion of this tethering region might be to overcome the that BX-C may have special properties which interfere action of the Fab boundaries, perhaps through contacts with the insulating activity of the gypsy element in this with promoter targeting sequences (PTS; Zhou and particular tissue. Since scs min is able to block PS10- and Levine, 1999) in each cis-regulatory domain. In this PS11-specific Abd-B expression in the CNS (Figure 2B), model, gypsy and scs min insulators would differ from the it would appear that, at least in this tissue, these two Fab boundaries because they disrupt this interaction insulators function differently or use distinct cofactors. mechanism, potentially by interfering with the PTS. An Our results demonstrate that like Fab-7, both gypsy alternative hypothesis comes from the recent finding and scs min can ensure the functional autonomy of the that when two copies of the gypsy insulator, instead of BX-C iab-6 and iab-7 cis-regulatory domains. However, a single one, are inserted between an enhancer and a these two elements differ from Fab-7 and other BX-C promoter, the insulator activity is neutralized (Muravyova boundaries such as Fab-8 in one critical respect they et al., 2001; Cai and Shen, 2001). The iab-7 cisboundaries also insulate the Abd-B promoter from the more distal regulatory domain is flanked by Fab-7 and Fab-8 boundaries. iab-5 and iab-6 cis-regulatory domains. Two models It is possible that like the gypsy insulator, the could potentially account for this critical difference. The Fab-7 and Fab-8 boundaries can pair and neutralize first postulates that there is some special mechanism each other, enabling the more distant iab-5 or iab-6 cis- in BX-C to facilitate long-distance interactions between regulatory domains to contact the Abd-B promoter. In

6 Molecular Cell 1150 Figure 4. The Su(Hw) but Not Mod Is Required for gypsy Insulation in the Context of Fab-7 A wild-type male has six abdominal segments. The seventh abdominal segment (A7), present in larvae, is suppressed during metamorphosis. In Fab-7 2, iab-7 is ectopically activated in most cells of A6. As a consequence, A6 assumes A7 identity, and most of the sixth tergite and sternite are absent (data not shown). Fab-7 2 is dominant, and these transformations can also be seen in heterozygous animals (Fab-7 2 / ). However, the homeotic transformation is weaker because only one dose of iab-7 is ectopically activated in A6. This is revealed by the smaller size of the sixth tergite (shown by two long arrows) and the absence of the sixth sternite (framed by four small arrows). In heterozygous males in which Fab-7 is substituted by the gypsy insulator, the sizes of the sixth tergite and sternite appear normal, indicating that gypsy can revert the Fab-7 2 phenotype. In the context of Fab-7, we have confirmed that insulation by gypsy requires Su(Hw). The male cuticle shown is heterozygous for the chromosome in which Fab-7 is replaced by the gypsy insulator and transheterozygote for a viable combination of su(hw) v / su(hw) f. Note that the phenotype is similar to Fab-7 2 /. In some but not all cases, the gypsy insulator requires a second protein, Mod(mdg4), for enhancer blocking (Gerasimova et al., 1995). In the homozygous mod(mdg4) u1 (mod u1 ) mutant male shown, both copies of Fab-7 have been replaced by the gypsy insulator. Note that the cuticle phenotype is the same as in Figure 3 (gypsy/gypsy), indicating that Mod is dispensable for insulating activity in this context. this model, Fab-8 would be unable to neutralize the events were scored for the excision of the bluetail transposon and insulator activity of either gypsy or scs. checked by PCR. On average, we recovered about 4 5 conversion events per 80 established lines. Experimental Procedures DNA Techniques, Fly Work, and Antibody Staining Gene Conversion As described in Mihaly et al. (1997). In order to substitute Fab-7 by the scs or gypsy insulators, we used gene conversion (Gloor et al., 1991). The precise scheme is Acknowledgments described elsewhere (Hogga and Karch, 1995). The fragment used as an ectopic template is indicated in Figure 1B. Note that it carries We thank Henrik Gyurkovics, Dale Dorsett, and Paul Schedl for the Fab-7 2 deletion. The gypsy or scs insulator (drawn at the same helpful discussions and Rakesh Mishra for critical reading of the scale) is inserted 140 bp to the left of the Fab-7 2 deletion, into an manuscript. We also thank Sue Celniker and Ed Lewis for providing Nsi1 site. For gypsy, we used a 340 bp fragment carrying the 12 the Abd-B antibody and iab-6 11 as well as Victor Corces for sending binding sites for the Su(Hw) protein (Geyer and Corces, 1992). In the gypsy insulator DNA clone. I.H. has been supported by grants the case of scs, we used a minimal 940 bp fragment (scs min ) known from the HFSPO and the Swiss National Foundation for Research. to have full insulating activity (Vazquez and Schedl, 1994). The inser- J.M. and S.B. have been supported by the Swiss National Foundation site of the bluetail transposon used to create the gap in Fab-7 tion for Research and by the state of Geneva. is also indicated (Galloni et al., 1993). In order to increase the gene conversion frequency, we recombined the P element carrying the ectopic template onto the bluetail chromosome. Putative conversion Received May 10, 2001; revised September 4, 2001.

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