A Comparison of Deformed-Responsive Elements

Regulation by Homeoproteins: A Comparison of Deformed-Responsive Elements

Jeffrey A. Pederson,*,1 James W. LaFollette,* Cornelius Gross,†,2 Alexey Veraksa,† William McGinnis† and James W. Mahaffey* *Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695-7614 and †Department of Biology, University of California, San Diego, California 92093 Manuscript received March 8, 2000 Accepted for publication June 1, 2000

ABSTRACT Homeotic genes of Drosophila melanogaster encode transcription factors that specify segment identity by activating the appropriate set of target genes required to produce segment-specific characteristics. Advances in understanding target gene selection have been hampered by the lack of genes known to be directly regulated by the HOM-C proteins. Here we present evidence that the gene 1.28 is likely to be a direct target of Deformed in the maxillary segment. We identified a 664-bp Deformed Response Element (1.28 DRE) that directs maxillary-specific expression of a reporter gene in transgenic embryos. The 1.28 DRE contains in vitro binding sites for Deformed and DEAF-1. The Deformed binding sites do not have the consensus sequence for cooperative binding with the cofactor Extradenticle, and we do not detect cooperative binding to these sites, though we cannot rule out an independent role for Extradenticle. Removing the four Deformed binding sites renders the 1.28 DRE inactive in vivo, demonstrating that these sites are necessary for activation of this enhancer element, and supporting the proposition that 1.28 is activated by Deformed. We show that the DEAF-1 binding region is not required for enhancer function. Comparisons of the 1.28 DRE with other known Deformed-responsive enhancers indicate that there are multiple ways to construct Deformed Response Elements.

ISTINCT morphological structures exist along the The products of the homeotic genes (homeoproteins) D anterior-posterior axes of animals. In Drosophila function as transcription factors (reviewed by Hayashi melanogaster the homeotic complex (HOM-C) genes are and Scott 1990). They contain a highly conserved 60- integral components of the pathways that ascribe differ- amino-acid DNA-binding domain known as the homeo- ent identities to cells along this axis (reviewed by domain (McGinnis et al. 1984; Scott and Weiner 1984; McGinnis and Krumlauf 1992). The HOM-C has been reviewed by Scott et al. 1989). Homeoproteins are conserved throughout evolution and is found in all ani- thought to specify segmental identity by activating the mals examined (McGinnis and Krumlauf 1992; Krum- appropriate battery of target genes that ultimately produce lauf 1994; Manak and Scott 1994). In Drosophila the segment-specific characteristics (reviewed by Andrew and HOM-C genes are found in two complexes: the Bithorax Scott 1992; Botas 1993; Morata 1993). Yet, how this complex and the Antennapedia complex (Lewis 1978; Ben- is achieved remains unknown. For several reasons it is der et al. 1983; Sanchez-Herrero et al. 1985; Akam unclear how the specificity of target gene selection is 1989; Kaufman et al. 1990; Kessel and Gruss 1990; attained. Different homeoproteins recognize very simi- Lufkin et al. 1992; Ramirez-Solis et al. 1993). The genes lar DNA sequences in vitro and have nearly identical of the Bithorax complex (Ultrabithorax, abdominal A, and binding affinities (Desplan et al. 1988; Hoey and Abdominal B) specify segment identity in the posterior Levine 1988; Affolter et al. 1990; Florence et al. 1991; thorax and abdominal region. The genes of the Anten- Dessain et al. 1992; Ekker et al. 1994; Walter et al. napedia complex (labial, proboscipedia, Deformed, Sex combs 1994). Further, a single homeoprotein can recognize a reduced, and Antennapedia) specify segmental identity in variety of DNA sequences (though almost all have the the head and anterior thorax. core sequence TAAT). On certain sites with TAAT (or subtle variants of that sequence) an immediately upstream site, TGAT, leads to cooperative heterodimer Corresponding author: James W. Mahaffey, Department of Genetics, binding of the Hox protein with the Extradenticle (Exd) North Carolina State University, Raleigh, NC 27695-7614. protein (Chan and Mann 1996). This interaction en- E-mail: [email protected] hances the sequence specificity of Hox DNA binding 1 Present address: Wyeth-Lederle Vaccines and Pediatrics, Sanford, NC 27330. and appears to be required for some Hox proteins to 2 Present address: Center for Neurobiology and Behavior, Columbia function as transcriptional activators (Li et al. 1999b). University, New York 10032. Hox/Exd heterodimer binding sites are found in a sub-

Genetics 156: 677–686 (October 2000) 678 J. A. Pederson et al. set of Hox response elements, but it is still unclear SmaI sites of pBS. The resulting construct was excised from whether Exd function is required on all response ele- pBS by digestion with BamHI and HindIII and subcloned into pHSS7, then into pHZ-white. Plasmids were purified using ments or only on some. QIAGEN’S (Valencia, CA) plasmid midi kit. DNA was ethanol To understand HOM-C specification of axial pat- precipitated and resuspended in injection buffer (Spradling terning, it is necessary to identify downstream target and Rubin 1982; Ashburner 1989) at a concentration of 400 genes that are controlled by specific homeoproteins. ng/␮l. Drosophila transformation followed the procedure of Identification of similarly regulated target genes would Robertson et al. (1988). Numbers of independent fly lines for each construct are as follows: 1.28 DRE,8;1.28 mut1-4,4; allow comparisons of the regulating enhancers, and 1.28 DRE Deformed binding region, 10; 1.28 DEAF-1 binding this could lead to the identification of important cues region, 3; chimera 1,5;chimera 2,3. in target gene regulation. There have been several DNase I footprint assays: Deformed protein was produced attempts to systematically identify downstream target in Escherichia coli and purified according to Dessain et al. genes (Gould et al. 1990; Gould and White 1992; (1992). DNase I footprinting experiments were carried out as described in Heberlein et al. (1985). DEAF-1 protein purifi- Wagner-Bernholz et al. 1991; Graba et al. 1992; cation and footprint reactions followed the protocol of Gross Mahaffey et al. 1993; Feinstein et al. 1995; Mastick and McGinnis (1996). et al. 1995; Botas and Auwers 1996). Unfortunately, Site-directed mutagenesis: Site-directed mutagenesis fol- the regulatory regions of only a few target genes have lowed the protocol of Kunkel et al. (1987). The 1.28 DRE was been characterized in sufficient detail to identify ho- subcloned into pBS as described above. The pBS clone was transformed into CJ236 and selected on ampicillin (Amp) and meotic response elements. These include the regulatory chloramphenicol (CAM). Cells from this culture were patched regions of teashirt (tsh), decapentaplegic (dpp), and De- onto LB plates containing 50 ␮g/ml Amp and 10 ␮g/ml CAM formed. tsh controls head vs. trunk development, and and were allowed to grow for 5 hr. A small loop of cells was high levels of tsh expression in the thoracic epidermis used to inoculate 10 ml 2ϫ YT containing 50 ␮g/mlAmp,10 require Antennapedia function (Fasano et al. 1991; ␮g/ml CAM, and 0.25 ␮g/ml uridine. After 40 min at 37Њ, the helper phage M13K07 was added. After an additional 30 McCormick et al. 1995). Transcriptional regulation of min, kanamycin was added to a final concentration of 70 ␮g/ tsh by Antennapedia is probably direct and involves se- ml and the culture was allowed to grow overnight at 37Њ. Phage quences in addition to the homeoprotein binding sites. were isolated by precipitation with 4% PEG/0.5 m NaCl. The dpp is a member of the transforming growth factor phage were resuspended in 100 ␮l TE, phenol-extracted, and (TGF)-␤ family of proteins and has been shown to be precipitated. A 10-fold molar excess of mutagenizing primer was added to template DNA. Primer extension reactions in- directly regulated by multiple homeoproteins in the em- cluded 5 units of Klenow, 0.5 mm dNTPs, and 800 units of bryonic midgut (Padgett et al. 1987, 1993; Capovilla T4 ligase. The primer extension reaction was transformed into et al. 1994; Manak et al. 1994; Sun et al. 1995). The SURE cells (Stratagene, La Jolla, CA). homeotic gene Deformed is itself a target of HOM-C re- In situ hybridization: Embryos for whole-mount in situ local- gulation through autoregulatory activation during em- ization of 1.28 or ␤-g␣l transcripts were dechorionated and bryonic development, and several autoregulatory ele- fixed following the procedure of Tautz and Pfeifle (1989). In situ hybridization analysis used ribonucleotide probes gen- ments have been identified that function as Deformed- erated with an RNA transcription kit (Stratagene) and DIG- responsive maxillary enhancers (Regulski et al. 1991; 11-UTP (Boehringer Mannheim, Indianapolis). Hybridization Zeng et al. 1994; Gross and McGinnis 1996). In addi- was carried out using modifications to the method of Tautz tion, two potential targets of Deformed have been iden- and Pfeifle (1989). Anti-DIG-AP (Boehringer Mannheim) tified, Distal-less (O’Hara et al. 1993) and 1.28 (Mahaf- was used to detect hybridization. Chimeric enhancer construction: The 120-bp module E ele- fey et al. 1993), respectively. ment was subcloned into the HindIII site of pBS and oriented In this article, we describe results of experiments indi- so the Deformed binding region could be amplified using cating that the Drosophila 1.28 gene is likely to be a the forward primer and the DEAF-1 binding region could be direct target of the Deformed homeoprotein. This amplified using the reverse primer. Primer 120 Deformed was allows us to compare activation of 1.28 with autoregula- designed to amplify the Deformed binding region of module E and has the sequence 5Ј-GGAAGCTTCGCCAGTCGGT tion of Deformed, where a molecular basis for De- TGG-3Ј. Primer 120 DEAF-1 was designed to amplify the formed regulation has been studied extensively. These DEAF-1 binding region and has the sequence 5Ј-GGAAGCTT comparisons provide evidence that activation of differ- GGGCACATTTCTT-3Ј. Each primer has a HindIII site added ent target genes by a single homeoprotein may involve to the end to simplify the subcloning of the fragments. PCR distinct pathways. was conducted using the following conditions: 95Њ for 40 sec, 52Њ for 1 min, 72Њ for 3 min for 25 cycles. Five microliters of each reaction was cut with 20 units of HindIII and subcloned MATERIALS AND METHODS into pBS. Positive clones were sequenced using a Sequenase kit (United States Biochemical, Cleveland) to confirm identity Transformation constructs: Genomic DNA fragments were and to ensure that no changes were incorporated during PCR. subcloned into pBluescript II KSϩ (pBS), then into pHSS7. For chimera 1, the 60-bp module E DEAF-1 binding region was This vector allows fragments to be excised with NotI ends, digested with HindIII and an aliquot of this digestion was which permits cloning into the NotI site of the transformation- ligated with the HindIII-digested Deformed binding region reporter vector pHZ-white. The 1.28 DRE was subcloned into of 1.28 in pBS and transformed into bacteria. Colonies were pBS in a three-part ligation. A 285-bp EcoRI fragment and a picked onto an LB/ampicillin grid plate, grown overnight, 379-bp EcoRI-BsrBI fragment were subcloned into the EcoRI- and transferred to Magna membrane (Micron Separations, Deformed Target Gene Selection 679

Inc., Westborough, MA) or Nytran (Schleicher & Schuelle; lary-specific expression of a ␤-gal reporter construct in Keene, NH). The filters were probed with module E DNA, transgenic flies. which was labeled using the Multiprime DNA labeling system (Amersham, Arlington Heights, IL) and [␣-32P]dCTP (New In the present study we examine portions of this 4.0- England Nuclear Life Science Products, Boston). The orienta- kb DNA fragment to identify smaller fragments con- tions of inserted fragments were determined via restriction taining maxillary enhancer elements. Fragments from digests. Chimera 2 was constructed similarly, except that the the 4.0-kb maxillary enhancer were inserted into the 60-bp module E Deformed binding region was subcloned as an P-element vector pHZ-white and tested for enhancer EcoRI-XhoI fragment adjacent to the DEAF-1 binding region of 1.28. activity in transgenic embryos. Multiple independent Isolation of P-element derivative lines: To mobilize the P transgenic fly lines were obtained for each construct element within the 1.28 gene, the 1.28 P chromosome was (see materials and methods). A 664-bp fragment re- crossed to a ␦ 2-3 transposase source following the method ferred to as the 1.28 DRE (1.28 Deformed Response Element) of Robertson et al. (1988). Two pairs of primers for PCR activates reporter expression in a manner similar to the were designed to detect deletions in adjacent 1.28 genomic DNA either 5Ј or 3Ј to the original P-element insertion point. entire 4.0-kb fragment. The position of this fragment Deletions were detected by lack of the specific PCR product with respect to the initial 4-kb enhancer is depicted in and verified by Southern blot analysis following standard mo- Figure 1. The 1.28 DRE directs ␤-gal expression in the lecular techniques (Sambrook et al. 1989). maxillary segment beginning at stage 14 (Figure 2C). Electrophoretic mobility shift assay with fragments of 1.28 ␤-Gal accumulation coincides with endogenous 1.28 ex- and module C: Probes for electrophoretic mobility shift assay were generated by PCR, gel-purified, cut with the enzyme AgeI, pression. Although the pattern of ␤-gal expression from and filled in with [32P]dCTP and Klenow. The 1.28 fragment the1.28 DRE is similar to the endogenous 1.28 gene, it corresponds to nucleotides 393–536 from Figure 4A. The mod- does not completely reproduce 1.28 gene expression. ule C fragment corresponds to nucleotides 680–805 from the The endogenous gene is expressed earlier and in more 2.7-kb Deformed epidermal autoregulatory element (Figure cells of the maxillary lobe. Maxillary expression by the 4; Zeng et al. 1994). 1.28 DRE is dependent upon the presence of functional 1.28 upper-strand primer: 5Ј-ACTACCGGTGCAGCGCTTC Deformed protein; ␤-gal is not observed in maxillary TTAGACTTTG; lobes of mutants lacking Deformed (data not shown). 1.28 bottom-strand primer: 5Ј-ACTACCGGTGCCTCAGCAA Like the endogenous gene, the 1.28 DRE directs expres- ACTAGCG; module C upper-strand primer: 5Ј-ATCACCGGTAAATTCG sion of the reporter in the developing gut beginning at AATTGAATTTTGGCGG; stage 12; this gut expression is independent of De- module C bottom-strand primer: 5Ј-ACTACCGGTCAAAA formed. TTTCACAAGATACAACGC. The 1.28 DRE contains in vitro Deformed binding Binding reactions (20 ␮l) were performed by incubating sites that are required for enhancer function in vivo: If labeled DNA fragments (80,000 cpm per lane) with protein Deformed regulation of 1.28 is direct, then the De- translation products in the binding buffer (Neuteboom and formed protein should bind to sequences within the Murre 1997), with 0.5 ␮g poly(dIdC) as a competitor. After 1.28 gene, and the binding sites should be required to 15 min of incubation at room temperature, complexes were enhance transcription. DNase I footprint analysis was resolved on a 5% polyacrylamide gel. Deformed and Exd proteins were produced using the TNT Quick Coupled in vitro performed to identify potential Deformed binding sites transcription/translation system (Promega, Madison, WI). De- within the 1.28 DRE, using full-length, bacterially ex- formed protein was made from pAR-Dfd (Jack et al. 1988), pressed Deformed protein (Regulski et al. 1991). Four and Exd was produced from pSP64ATG-Exd (van Dijk et al. Deformed binding regions (DBS1–4) were identified 1993). within the 1.28 DRE (Figure 3A); the sequences of the sites are indicated in Figure 4A. The sites all contain the core sequence ATTA/TAAT common to most HOM-C RESULTS protein binding sites. Identification of a 1.28 Deformed Response Element: To test whether these Deformed binding sites are Maxillary expression of 1.28 begins at about stage 10 of required for reporter activation in vivo, we used site- embryogenesis in the posterior lateral epidermis of the directed mutagenesis to change the ATTA/TAAT core maxillary lobes, continues through stage 16, and then and one base on either side to G-C-rich sequences at declines during later development (Figure 2, A and B; all four Deformed-binding sites. DNaseI footprinting see also Mahaffey et al. 1993). 1.28 is also expressed demonstrated that Deformed could no longer bind to in the gut and the anterior and posterior spiracles. We these sites (data not shown). The altered fragment (1.28 demonstrated previously that expression of 1.28 in the mut1-4) was cloned into pHZ-white, transformed into maxillary epidermis is Deformed dependent; embryos flies, and embryos containing this enhancer were as- lacking functional Deformed protein do not express sayed for ␤-gal accumulation. This 1.28 mut1-4 element 1.28 in the maxillary segment, though expression in does not direct expression of ␤-gal in maxillary cells of other regions of the embryo is not altered (Mahaffey stage 14 embryos as does the wild-type 1.28 DRE. We et al. 1993). Furthermore, we demonstrated that a 4.0- did notice some ␤-gal expression from the 1.28 mut1-4 kb fragment from the 1.28 gene was able to direct maxil- beginning at about stage 16, when a low level of ␤-gal 680 J. A. Pederson et al.

Figure 1.—Molecular map of the 1.28 DRE. The top map shows the initial 4-kb SalI fragment originally shown to direct expression in the maxillary segment. The enlargement shows the position of the 664-bp 1.28 DRE. The potential starts and direction of transcription are shown by arrows, and the insertion position of the enhancer trap P element is marked. Bases are numbered from the right EcoRI site to indicate the sub- fragments described in this study. could be detected in the maxillary epidermis (Figure formed (module A-F; Zeng et al. 1994), and one created 2D). This level of ␤-gal is signiﬁcantly lower than that by changing two bases within a labial response element so observed for the wild-type 1.28 DRE and is comparable that this element now responds to Deformed [repeat 3 to levels observed from the pHZ-white vector alone (Haerry and Gehring 1997; data not shown). We conclude that the sequences forming the in vitro binding sites are necessary for enhancer function in vivo. Further dissection of the 1.28 DRE: Several epidermal Deformed-responsive enhancers have been described, some identiﬁed within the autoregulation region of De-

Figure 2.—Comparison of 1.28 expression and ␤-gal expression generated by 1.28 DRE reporter constructs. Arrows Figure 3.—Deformed and DEAF-1 bind to the 1.28 DRE. point to the expression in the maxillary segment. (A and B) (A) DNase I footprint of the 1.28 DRE by bacterially expressed Expression of the endogenous 1.28 gene in the maxillary Deformed protein. G denotes sequencing lane. Increasing segment at stages 12 and 14 of embryogenesis, respectively. amounts of Deformed protein from left to right (0, 100, and (C) The 1.28 DRE directs ␤-gal expression in the maxillary 500 ng, respectively). Deformed binds to the four sites indi- segment beginning at stage 14 in a subset of endogenous 1.28- cated by bars. The sequence of each Deformed binding site expressing cells. (D) ␤-Gal expression from the 1.28 mut1-4 is shown in Figure 4. (B) DNaseI footprint of the 1.28 DRE enhancer in a stage 16 embryo. Expression from 1.28 mut1-4 by DEAF-1 protein. G denotes sequencing lane. Increasing is weak and delayed with respect to the 1.28 DRE. ␤-Gal expres- amounts of bacterial DEAF-1 protein from left to right (0, 1, sion detected by in situ hybridization with ␤-gal antisense and 5 ng, respectively). In each panel, the top strand is labeled. probe. In all ﬁgures, anterior is to the left. The sequence of the top strand is shown in Figure 4. Deformed Target Gene Selection 681

(TA); Chan et al. 1997]. Along with Deformed, there entirely within the 285-bp Deformed-binding portion is evidence for a role of several other proteins in activa- of the 1.28 DRE. tion of these enhancer elements. The DEAF-1 protein Exd functions as a cofactor at many Hox enhancers, was identified by its ability to bind to a region of the including some Deformed-responsive maxillary en- 120-bp Deformed autoregulatory enhancer, module E hancers. We used electrophoretic mobility shift assays (Zeng et al. 1994; Gross and McGinnis 1996). Also, to determine whether or not the Exd protein could the Extradenticle protein (Exd) has been shown to be bind to the 1.28 DRE (Figure 5). As a control, we chose required for activation of some Deformed-responsive module C from the 2.7-kb Deformed epidermal autoregu- maxillary enhancers (Chan et al. 1997; Pinsonneault latory enhancer (Zeng et al. 1994). Module C contains et al. 1997). Li et al. (1999a) have recently identified a a consensus Deformed/Exd heterodimer binding site sequence found at several Deformed-responsive maxil- (TGATTAAT). A fragment containing this region from lary enhancers that likely forms binding sites for un- module C binds Deformed alone, and a trimeric complex known factors that are required for activation of these between Deformed, Exd, and the module C DNA probe enhancers in vivo. This sequence was first identified is observed upon addition of Exd to the binding reac- within an imperfect inverted region of the module E tion. No trimeric complex was observed in lanes with enhancer. Since expression of 1.28 and autoregulation Deformed, Exd, and the 1.28 DRE probe, though De- of Deformed occur in similar cells of the maxillary epider- formed protein could form a stable complex with the mis and at a similar stage of embryogenesis, it is possible 1.28 DRE (Figure 5). We did not detect binding of Exd that one or more of these factors contributes to 1.28 to the 1.28 DRE either alone or as a complex with De- enhancer function. We next investigated whether any formed. of these factors might be involved in activation of the Other yet-unknown factors are likely to be involved 1.28 DRE. as partners with Hox proteins in target gene activation. We first looked for similarities between the 1.28 DRE These factors would have binding sites that are required and the Deformed autoregulation enhancer. The DEAF-1 at specific enhancers. A candidate for such a binding protein has been shown to bind to module E, where there site is present in an imperfect inverted repeat sequence are several binding sites for this protein. Using DNaseI found in the Deformed autoregulatory enhancer, module E (Zeng et al. 1994; Gross and McGinnis 1996; footprint analysis, we determined that a 300-bp region Li et al. 1999a). A similar imperfect inverted repeat within the 1.28 DRE contains eight DEAF-1 binding sites sequence is located within the 1.28 DRE (see Figure 4, (Figure 3B). The sequences of the regions protected A and B). Since this inverted repeat is located within from DNaseI digestion by DEAF-1 are indicated in Fig- the DEAF-1 binding region, it was removed from the ure 4A. These binding sites ranged from 10 to 53 bp in 1.28 DRE in the experiments described above, indicat- length and include the imperfect inverted repeat. The ing that this region does not participate in activating larger protected region likely contains multiple binding the 1.28 DRE in reporter constructs. sites for the DEAF-1 protein. It is interesting to note The 1.28 gene was originally identified in an enhancer that the Deformed and DEAF-1 binding sites do not trap screen (Mahaffey et al. 1993). In this enhancer overlap. The Deformed binding sites are located within trap line the P element is inserted into the DEAF-1 Ј the 3 half of this enhancer, and the DEAF-1 binding binding region of the endogenous 1.28 DRE (see Ј sites lie within the 5 half. P-element insertion site in Figures 1 and 4). Insertion The 1.28 DRE can be divided at an EcoRI restriction of this element causes a significant reduction in 1.28 site to give a 285-bp fragment containing the Deformed expression in the maxillary segment (Figure 6B). We binding sites and a 379-bp fragment with the DEAF-1 mobilized the P element to create deletions in adjacent region (Figures 1 and 4A). We tested these two separate genomic DNA (Robertson et al. 1988). P-element-deriv- regions for enhancer function in vivo by cloning them ative lines were screened by PCR and Southern blot into pHZ-white, transforming them into flies, and stain- analysis to identify lines where mobilization of the P ing embryos to detect ␤-gal. The 285-bp fragment con- element created deletions in the region upstream of taining the four Deformed binding sites is sufficient to the 1.28 gene but not extending into the transcribed kb upstream 1ف direct maxillary expression of the reporter gene in vivo, portion of the gene. A deletion of and this expression is indistinguishable from the com- of the original P-element insertion site was created in plete 1.28 DRE (data not shown). Conversely, the 379- derivative line 13 (data not shown). This deletion re- bp fragment containing the DEAF-1 binding region moves the two DEAF-1 binding regions upstream of does not direct maxillary expression of the reporter the P-element insertion site, including the imperfect gene at any stage; therefore, this fragment repressed inverted repeat (see Figure 4 for the positions of these the stage 16, weak, background expression usually seen sites). Expression of the 1.28 in wild type and line 13 with the pHZ-white vector alone (data not shown). We embryos is indistinguishable (Figure 6, A and C, respec- conclude that the information needed to direct maxil- tively). This further indicates that the imperfect inverted lary expression during stages 14 and 15 is contained repeat is not required for the expression of 1.28. 682 J. A. Pederson et al.

Figure 4.—(A) Sequence of the 1.28 DRE. The Deformed binding sites are shown as boxes and are marked DBS1–4. DEAF-1 binding sites are underlined, with the bases forming the imperfect inverted repeat marked with an asterisk. Two potential transcription start sites are indicated by arrows with “start” written above them. The P-element insertion point is shown. Note that the De- formed and DEAF-1 binding sites do not overlap. The four Deformed binding sites are within the EcoRI restriction fragment, and the eight DEAF-1 binding sites are located within the BsrBI-EcoRI restriction fragment. (B) Alignment of the module E (top) and 1.28 DRE (bottom) inverted repeat sequences. Identical bases are marked with an an asterisk. The sequence identiﬁed as necessary for expression of module E by Li et al. (1999a) is shown in boldface type. Note that this sequence is not found in the inverted repeat region nor else- where in the 1.28 DRE.

Further comparisons of the 1.28 DRE and module E: ble for the differences in enhancer strength, that is, in The overall composition of binding sites making up the the timing and number of cells expressing the reporter. 1.28 DRE and module E is quite similar. Both enhan- Chimera 1 was created so that the 1.28 Deformed bind- cers contain nonoverlapping Deformed and DEAF-1 ing sites were fused to regions 5 and 6 of module E, binding regions. Both enhancers function similarly in which contain DEAF-1 binding sites (Figure 7 C). When vivo, but module E directs reporter expression earlier assayed after transformation, chimera 1 functions as a and in more cells of the maxillary segment than does the maxillary enhancer and drives expression at levels com- 1.28 DRE (Figure 8, A and B, respectively). To determine parable to module E (Figure 8, C and A, respectively). whether the differences are attributable to speciﬁc re- Expression of the reporter was earlier, at higher levels, gions of these enhancers, we constructed chimeric en- and in a few more cells than the intact 1.28 DRE. Chimera hancers composed of portions of each. Maps of the 2 had the 1.28 DEAF-1 binding region linked 5Ј to the chimeras are shown in Figure 7. Using these chimeric Deformed-binding portion of module E (Figure 7D). In enhancers we could address whether the Deformed and contrast to chimera 1, chimera 2 did not activate maxillary DEAF-1-binding regions of the two enhancers are simi- expression at any stage (Figure 8D). Furthermore, the lar in function and ascertain which regions are responsi- 1.28 DRE DEAF-1 binding region again appears to re- Deformed Target Gene Selection 683

Figure 5.—Deformed/Exd protein binding to Hox binding sites in the 1.28 element. Electrophoretic mobility shift assays were performed as described in materials and methods. Deformed protein alone formed a stable complex with the 1.28 DRE fragment that in- cluded all Deformed binding sites iden- tiﬁed in the 1.28 DRE. This binding was not enhanced by addition of Exd. A fragment of the 2.7-kb Deformed epidermal autoregulatory enhancer module C (Zeng et al. 1994), which contains a consensus Deformed/Exd heterodimer binding site (TGATTAAT), was bound by De- formed alone with a slightly higher af- ﬁnity than the 1.28 fragment. Upon addition of Exd to the binding reaction, a trimeric complex among Deformed, Exd, and the module C DNA probe was observed (indicated by arrow). A trimeric complex was not observed in lanes with Deformed, Exd, and the 1.28 DRE DNA probe. Control lysate was added to the binding reactions to keep the total lysate volume in each reaction at 2 ␮l.

press even the slight background expression observed genic ﬂies (Mahaffey et al. 1993). In the present study with pHZ-white. These results indicate that the De- we examined fragments of this 4.0-kb DNA element formed and DEAF-1 binding regions of the two en- to identify smaller maxillary enhancer elements. We hancers are not identical. module E appears to contain identiﬁed a 664-bp DNA fragment, encompassing the a site or sequence that increases enhancer activity when 1.28 start of transcription and containing in vitro bind- fused to other Deformed binding sites. This result sup- ing sites for Deformed, that functions as a maxillary ports the conclusion of Li et al. (1999a), that a sequence enhancing maxillary expression is present in this fragment. Furthermore, this demonstrates that the function can be transferred to another response element.

DISCUSSION We previously demonstrated that a 4.0-kb fragment from the 1.28 gene was able to direct maxillary-speciﬁc expression of ␤-gal from a reporter construct in trans-

Figure 6.—The imperfect inverted repeat is not required Figure 7.—Schematic drawing of chimeric enhancer trans- for in vivo 1.28 expression in the maxillary segment. All em- formation constructs. Deformed binding sites within the 1.28 bryos are about stage 12. (A) 1.28 mRNA expression in the DRE are indicated by solid boxes and the module E Deformed maxillary segment of a wild-type embryo. (B) 1.28 mRNA binding site is shown as an open box. The DEAF-1 binding expression in the maxillary segment of the enhancer trap region of the 1.28 DRE is indicated by a solid oval. The module E line 1.28P. Note that the expression is signiﬁcantly reduced DEAF-1 binding region is indicated with an open oval. Arrows compared to wild-type 1.28 expression. (C) In P-element deriv- denote orientation of DNA fragments. Restriction sites are as ative line 13, 1.28 mRNA expression is indistinguishable from follows: B, BsrBI; H, HindIII; R, EcoRI; X, XhoI. H* indicates a the wild-type expression pattern. HindIII site generated by PCR (see materials and methods). 684 J. A. Pederson et al.

tween the 1.28 DRE and other Deformed response elements does not appear to be the number of Deformed binding sites within each enhancer, since multimeriza- tion of module E, such that there are four Deformed binding sites, does not eliminate the requirement for additional regulatory elements. From the lack of heterodimer consensus sites and from our protein-binding studies, we suspect that activation of the 1.28 DRE does not require binding of the Exd cofactor as a heterodimer with Deformed, though we cannot rule out an independent role for Exd in activating this element. Li et al. (1999b) suggest that the interaction between Exd and Deformed at a heterodimer binding site leads to exposure of the transcriptional activation domain of the Deformed protein and thereby activation of the target gene. If in fact the 1.28 DRE can activate expression without Exd, then either some other unidentified factor must lead to exposure of this activation domain, or perhaps certain arrange- ments of Deformed binding sites can alleviate this re- Figure 8.—Comparison between Deformed response ele- quirement. In some cases Exd may function when not in ments and chimeric enhancers. All embryos are about stage 14 and arrows indicate maxillary expression. (A) Maxillary the heterodimer Hox/Exd arrangement. The Deformed expression of ␤-gal directed by module E. (B) 1.28 DRE gener- module E enhancer does not contain the consensus ates maxillary ␤-gal expression at a lower level compared to TGATTAAT Exd/Deformed binding sequence, but an module E. (C) Chimera 1 directs ␤-gal expression in the maxil- Exd binding site is located several bases from the De- lary segment at higher levels compared to the 1.28 DRE. (D) formed binding site. The sequence forming this Exd Chimera 2 does not express ␤-gal at any stage. ␤-Gal expression was detected by RNA in situ hybridization. binding site appears to be important at some level be- cause eliminating this sequence reduces maxillary expression of a reporter gene, though maxillary expres- enhancer. That the sequences forming the in vitro De- sion is not eliminated (Pinsonneault et al. 1997). At formed binding sites are required for enhancer function this time, we do not know if such an independent role in vivo supports our proposition that the 1.28 gene may for Exd is needed to activate the 1.28 gene. Regulation be a direct target of Deformed in the maxillary segment. of the endogenous gene is complex and likely requires However, we cannot absolutely rule out that one or other regulatory factors and binding sites. It is possible, more of these sites may be interacting with other factors if not likely, that there are other maxillary enhancer in vivo. elements at the 1.28 gene. Activation of 1.28 and autoregulation of Deformed oc- As mentioned above, to function as a maxillary en- cur simultaneously in many cells along the posterior- hancer module E requires at least one sequence in addi- lateral edge of the maxillary segment beginning at about tion to the Deformed and Exd binding sites. This se- stage 10. This suggests that common factors may play quence is found in an imperfect inverted repeat (Zeng roles in activating these two genes. Clearly, Deformed et al. 1994; Gross and McGinnis 1996; Li et al. 1999a). is likely a component of both pathways, since Deformed Site-directed mutagenesis of this imperfect inverted re- is required for 1.28 expression and autoregulation of peat abolishes module E enhancer function. Though it Deformed (Bergson and McGinnis 1990; Mahaffey et seemed noteworthy that a similar imperfect inverted al. 1993; Zeng et al. 1994). However, though Deformed repeat sequence is located within the 1.28 DRE (Figure binds to sites within the 1.28 DRE and the Deformed 4B), that sequence is not required for 1.28 DRE en- autoregulatory enhancer module E (Zeng et al. 1994), hancer function, and deletion of this repeat has no the enhancers are not equivalent. The sequences form- consequence on expression of the endogenous 1.28 ing the Deformed-binding region of module E will not gene. Attaching the module E inverted repeat sequence function as a maxillary enhancer without the addition to the Deformed binding portion of the 1.28 DRE does of other elements. This is similar to the repeat 3[TA] increase activity of the 1.28 DRE, indicating that this maxillary enhancer as this enhancer requires Deformed sequence can function in a heterologous enhancer. We and Exd binding sites, and binding to each half-site is favor the idea that the module E inverted repeat region required for maxillary expression of the reporter (Chan contains a binding site or sites for other unknown fac- et al. 1997). In the 1.28 DRE, it appears that the 285-bp tors, and that these factors act to enhance maxillary- Deformed-binding region is sufficient to direct maxil- specific expression. Li et al. (1999a) have shown that lary expression of a reporter gene. The difference be- such a site is likely to be within the inverted repeat Deformed Target Gene Selection 685 sequence of module E. They propose that factors bind and the Bithorax-complex in Drosophila melanogaster. J. Mol. Biol. 168: 17–33. to sequences GGC and AAAGC of the module E repeat. Bergson, C., and W. McGinnis, 1990 An autoregulatory enhancer This sequence is not present in the 1.28 DRE, suggesting element of the Drosophila homeotic gene Deformed. 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