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Transcriptional regulation of a pair-rule stripe in Drosophila S t e p h e n S m a l l , 1 R a c h e l Kraut, 1 T i m o t h y H o e y , 2 R a h u l W a r r i o r , 3 a n d M i c h a e l L e v i n e 1 D e p a r t m e n t of Biological Sciences, F a i r c h i l d C e n t e r , C o l u m b i a U n i v e r s i t y , N e w York, N e w Y o r k 10027 U S A

The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is i n i t i a t e d by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd} and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Kriippel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a c o m p e t i t i o n or short-range quenching mechanism, whereby the binding of gt and K r interferes with the binding or activity of b c d and h b activators at overlapping or neighboring sites within the eve stripe 2 p r o m o t e r element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements. [Key Words: Pair-rule gene; Drosophila; p a t t e r n formation; stripe; short-range repression] Received January 16, 1991; revised version accepted March 5, 1991.

Previous genetic screens have identified - 5 0 regulatory genes t h a t control early development in Drosophila, and approximately half of these subdivide the e m b r y o into a repeating series of segments (Lewis 1978; Kaufman et al. 1980; Nfisslein-Volhard and Wieschaus 1980). M a n y of t h e s e g m e n t a t i o n genes have been cloned and character- ized (e.g., Laughon and Scott 1984; M c G i n n i s et al. 1984), and the vast m a j o r i t y encode nuclear factors con- taining well-characterized DNA-binding motifs, includ- ing the h o m e o domain (Levine and Hoey 1988; Scott et al. 1989), zinc fingers (Rhodes and Klug 1988), t h e h e l i x - loop--helix (Murre et al. 19891, and t h e leucine zipper (Vinson et al. 1989). Each of these genes shows a u n i q u e pattern of expression in t h e early embryo and is active in a specific subset of cells (Akam 1987; Ingham 1988). It has been s h o w n in n u m e r o u s instances t h a t the misex- pression of a particular s e g m e n t a t i o n gene causes disrup- tions i n t h e p a t t e r n of t h e embryo~ s o m e t i m e s , these phenotypes m i m i c other s e g m e n t a t i o n m u t a n t s (e.g., Struhl 1985).

Spatially restricted patterns of s e g m e n t a t i o n gene ex- pression depend on a hierarchic series of gene interac- tions. T h e first step in this hierarchy is the establish- m e n t of crude gradients of m a t e r n a l morphogens in un- fertilized eggs and early embryos. A m o n g these is the h o m e o box protein bicoid (bcd), w h i c h plays a k e y role

Present addresses: 1Biology Department, Bonnet Hall, University of Cal- ifornia, San Diego, La Jolla, California 92093-0346 USA; 2Department of Molecular and Cell Biology, University of California, Berkeley, Califor- nia 94720 USA; 3Roche Institute of Molecular Biology, Nutley, New Jersey 07110 USA.

in the i n i t i a t i o n of gap gene expression {Berleth et al. 1988; Driever and Nfisslein-Volhard 1988}. Each of the five k n o w n gap genes is expressed in one or two broad domains t h a t span several adjacent segment primordia (e.g., Gaul and J~ickle 1987; Pignoni et al. 1990}. T h e y are t h o u g h t to control s e g m e n t a t i o n p r i m a r i l y t h r o u g h t h e regulation of t h e pair-rule genes which, in turn, i n i t i a t e the expression of - 1 0 different s e g m e n t polarity genes (for review, see Ingham 1988}. T h i s regulatory cascade occurs q u i t e rapidly and c u l m i n a t e s just 5 hr after fertil- ization in t h e precise expression of t h e s e g m e n t polarity genes w i t h i n t h e l i m i t s of single cells in every s e g m e n t p r i m o r d i u m (e.g., DiNardo et al. 1985; Kornberg et al. 1985; Hooper and Scott 1989; N a k a n o et al. 1989}.

A recurring t h e m e of the s e g m e n t a t i o n h i e r a r c h y is the progressive r e f i n e m e n t in t h e p a t t e r n s of gene ex- pression. A t every step in the h i e r a r c h y a given segmen- tation gene m a k e s a relatively sharp "on/off" choice in response to more crudely distributed regulatory prod- ucts. Here, we present evidence t h a t this r e f i n e m e n t in expression involves t h e i n t e r a c t i o n s of distinctive com- binations and c o n c e n t r a t i o n s of regulatory factors w i t h promoter sequences t h a t have t h e properties of an on/off switch. In particular, we have e x a m i n e d t h e i n t e r a c t i o n of t h e gap genes w i t h the pair-rule gene even-skipped (eve).

eve encodes a h o m e o box protein t h a t is expressed in a series of seven transverse stripes along the length of t h e embryo t h a t play a k e y regulatory role in t h e establish- m e n t of t h e m e t a m e r i c body plan (Harding et al. 1986; Macdonald et al. 1986; Frasch et al. 1987). T h e f o r m a t i o n

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of the striped expression p a t t e r n involves a two-step pro- cess. First, it has been s h o w n by p r o m o t e r fusion studies t h a t separate cis p r o m o t e r e l e m e n t s direct the initial ex- pression of individual stripes (spanning five to six nu- clei). There is considerable evidence t h a t this initial seven-stripe p a t t e r n is controlled by the broadly distrib- uted, overlapping domains of the gap gene products. Mu- tations in a n y of the gap genes lead to a severe disruption of the initial seven-stripe p a t t e r n (Frasch and Levine 1987; Driever and Nfisslein-Volhard 1988; M. Frasch, unpubl.). Later, the e v e protein present w i t h i n these ini- tially broad stripes interacts w i t h a distal enhancer ele- m e n t located between - 5.9 and - 5.2 kb u p s t r e a m from the transcription start site (Goto et al. 1989; Harding et al. 1989; Jiang et al. 1991), w h i c h helps refine the stripes so t h a t each spans just two to three cells and shows a n t e r i o r - p o s t e r i o r polarity by the onset of gastrulation (Lawrence et al. 1987; Frasch et al. 1988).

We present a model for the transcriptional regulation of the i n i t i a t i o n of eve stripe 2. We have focused on this stripe because previous p r o m o t e r fusion analyses and ge- netic studies have provided considerable information about the cis and trans c o m p o n e n t s t h a t participate in its localized expression. A t r u n c a t e d p r o m o t e r contain- ing 1.7 kb of e v e 5'-flanking sequence is sufficient to drive t h e expression of a reporter gene (lacZ) w i t h i n the l i m i t s of stripe 2 (Goto et al. 1989; Harding et al. 1989). A 480-bp internal deletion between - 1 . 6 and - 1 . 1 kb abolishes expression (Goto et al. 1989). Potential trans- regulators of stripe 2 have been identified on the basis of examining the distribution of eve protein in all k n o w n s e g m e n t a t i o n m u t a n t s (Frasch and Levine 1987; Driever and Nfisslein-Volhard 1988; M. Frasch, unpubl.). These studies, as well as the tight linkage of the wild-type ex- pression patterns, suggest t h a t the gap genes h u n c h b a c k (hb), g i a n t (gt), and Krfippel (Kr) are the m o s t likely can- didates for regulators of stripe 2 expression. There is ev- idence t h a t two of these, h b and Kr, directly regulate the expression of the stripe because t h e y bind to closely linked sites in the stripe 2 p r o m o t e r e l e m e n t (Stanojevic et al. 1989). Here, we show t h a t the leucine zipper pro- tein gt (E. Eldon and V. Pirrotta, pers. comm.) binds to stripe 2 sequences as well. Surprisingly, we also found several binding sites for the m a t e r n a l morphogen bcd. T h e gt- and Kr-binding sites overlap or are closely linked to the h b and b c d sites. T r a n s i e n t cotransfection assays suggest t h a t b c d and h b activate transcription by binding to stripe 2 sequences. This activation is repressed by the coexpression of either Kr or gt, and D N A binding is re- quired for repression by either protein, suggesting t h a t gt and Kr repress expression through a c o m p e t i t i o n or short-range quenching m e c h a n i s m . We discuss the im- plications of such short-range repression w i t h respect to the evolution of complex promoters.

R e s u l t s

A m o d e l for t h e g e n e t i c c o n t r o l of eve expression at stripe 2

T h e wild-type l i m i t s of h b (Tautz et al. 1987), Kr (Rosen-

berg et al. 1986), and gt (Mohler et al. 1989) expression suggest t h a t t h e y have a direct role in regulating the initiation of e v e expression w i t h i n the l i m i t s of stripe 2 (Fig. 1 A-C). T h e anterior domain of h b expression com- pletely overlaps w i t h e v e stripe 2 (Fig. 1A; Stanojevic et al. 1989; Warrior and Levine 1990). b c d is expressed in a gradient t h a t extends beyond the anterior h b domain (Driever and Nfisslein-Volhard 1988) and encompasses the region of the stripe. The anterior l i m i t of the Kr pat- tern abuts the posterior border of stripe 2 (Stanojevic et al. 1989; Fig. 1B), while gt abuts t h e anterior border of the stripe (Fig. 1C).

The expression patterns for the four putative regula- tors of stripe 2 are s u m m a r i z e d in Figure 1D. In this model, the m a t e r n a l morphogen b c d and the gap gene h b activate eve expression in the stripe 2 region, and the borders of the stripe are formed by repressive interac- tions by gt anteriorly and Kr posteriorly. This model is supported by previous studies of e v e expression patterns in various m u t a n t s (Frasch and Levine 1987): (1) eve stripe 2 is greatly reduced or missing in h b - e m b r y o s ; (2) stripe 2 appears to be fused w i t h stripe 3 in K r - embryos; and (3) stripe 2 appears to be fused w i t h stripe 1 in g t - embryos. Furthermore, Goto et al. (1989) have shown t h a t stripes 2 and 3 are fused in K r - embryos carrying a heterologous p r o m o t e r construct t h a t selectively ex- presses stripes 2, 3, and 7.

These studies suggest t h a t the domain of expression in stripe 2 expands in g t - and K r - e m b r y o s but cannot rig- orously rule out the possibility t h a t the expansion de- rives from the adjacent stripes ( 1 in g t - and 3 in K r - ). To distinguish among these possibilities, we examined an e v e p r o m o t e r - l a c Z gene fusion (Fig. 2B) that expresses l a c Z only at the positions of stripes 2 and 7 (Fig. 2C). In K r - embryos the fusion gene is expressed in a broad band rather than a narrow stripe, due to an expansion of its posterior border (Fig. 2D). Similarly, gt + gene activity seems to be i m p o r t a n t for the specification of the ante- rior stripe border because there is an anterior expansion of I a c Z expression in g t - embryos (Fig. 2E). Stripe 2 ex- pression is lost or greatly reduced in b c d - and h b - em- bryos, suggesting t h a t these genes exert a positive effect on its expression (data not shown). We have n o t exam- ined the expression of the stripe 2 - I a c Z gene fusion in a n y other s e g m e n t a t i o n m u t a n t s , because the earlier studies (Frasch and Levine 1987) indicated t h a t none of the other genes were required for the initiation of stripe 2 expression. For example, stripe 2 appears normal in k n i r p s and tailless m u t a n t s , as well as in maternal mu- tants that disrupt the torso or n a n o s morphogenetic or- ganizing centers.

D N A - b i n d i n g assays

T h e type of genetic studies described above fail to dis- tinguish between direct and indirect regulatory interac- tions. As a first step toward d e t e r m i n i n g w h e t h e r the interactions m i g h t be direct, we performed DNA-bind- ing assays using stripe 2 sequences and bed, h b , gt, and Kr proteins. Previous studies have identified a n u m b e r of

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eve stripe 2

F i g u r e 1. Wild-type expression patterns of putative regula- tors of eve stripe 2. {A). Wild-type embryo stained with a mixture of eve (red) and h b (green) antibodies (regions of over- lap appear yellow). This embryo and the other embryos shown here and in Fig. 2 are oriented with anterior to the l e f t and dorsal up. The anterior domain of h b extends to the posterior border of stripe 3. The posterior h b domain overlaps the sev- enth eve stripe. (B) Wild-type embryo stained with a mixture of eve (red) and Kr (green) antibodies. The broad central do- main of Kr expression extends from the posterior border of eve stripe 2 to the anterior border of stripe 5. (C) Wild-type em- bryo stained with a mixture of eve (green) and gt (red) anti- bodies. The anterior domain of gt expression extends to the anterior limit of stripe 2. The posterior gt domain extends from the posterior border of stripe 5 to the anterior border of stripe 7 (see also Mohler et al. 1989. (D) Summary of the trans regulation of eve stripe 2. The approximate expression pat- terns of the b c d and h b activators are represented by heavy black curves. Together, these proteins coincide with a broad domain where the stripe 2 element can be activated. The borders of the stripe (represented by the vertical lines at the b o t t o m ) are established by repressive interactions mediated by gt (anterior border) and Kr (posterior border).

h b - a n d K r - b i n d i n g sites (Stanojevic et al. 1989). In t h i s s t u d y w e h a v e o v e r e x p r e s s e d full-length, n o n f u s i o n b c d a n d g t p r o t e i n s in E c h e r i c h i a c o l i (Studier a n d M o f f a t t 1986) a n d u s e d t h e s e to p e r f o r m s y s t e m a t i c D N a s e I foot- p r i n t a s s a y s across t h e e v e s t r i p e 2 e l e m e n t . W e f o c u s e d p r i m a r i l y on p r o m o t e r s e q u e n c e s e x t e n d i n g f r o m a b o u t - 1 7 0 0 to - 8 0 0 bp b e c a u s e p r e v i o u s s t u d i e s s u g g e s t e d t h a t t h i s r e g i o n p l a y s a p a r t i c u l a r l y i m p o r t a n t role in stripe 2 e x p r e s s i o n (Goto et al. 1989). F u r t h e r m o r e , stud- ies on h b a n d K r i n d i c a t e a l a c k of b i n d i n g sites in prox- i m a l r e g i o n s of t h e p r o m o t e r (Stanojevic et al. 1989). Ex- a m p l e s of t h e b i n d i n g e x p e r i m e n t s are p r e s e n t e d in Fig- ure 3.

Figure 3 A s h o w s t h e r e s u l t s of a D N a s e I p r o t e c t i o n assay, u s i n g a f f i n i t y - p u r i f i e d b c d p r o t e i n a n d a - 4 0 6 - b p

D r a I I I - B a l I D N A f r a g m e n t , w h i c h e x t e n d s f r o m a b o u t - 1.3 kb to - 900 bp u p s t r e a m f r o m t h e s t a r t site. S t r o n g p r o t e c t i o n is o b s e r v e d for t h e s e q u e n c e T C G A A G G - G A T T A G G l o c a t e d at a b o u t - 1285 bp, w h i c h i n c l u d e s a n 8 o u t of 9 m a t c h w i t h t h e b c d core c o n s e n s u s se- q u e n c e ( G G G A T T A G A ) d e t e r m i n e d b y D r i e v e r a n d N f i s s l e i n - V o l h a r d (1989). A s e c o n d b c d - b i n d i n g site is s e e n at a b o u t - 1190 bp a n d also c o n t a i n s a n 8/9 m a t c h w i t h t h e c o n s e n s u s ( G G G A T T A G C ) .

Figure 3B s h o w s t h e D N A - b i n d i n g a c t i v i t y of t h e g t protein. In t h i s e x p e r i m e n t a 3 4 9 - b p R s a I - S t y I D N A frag- m e n t (located b e t w e e n a p p r o x i m a t e l y - 1 . 5 a n d - 1 . 2 kb) w a s 3~P-labeled o n t h e n o n c o d i n g s t r a n d a n d i n c u - b a t e d w i t h i n c r e a s i n g a m o u n t s of t h e g t p r o t e i n . Se- q u e n c e s t u d i e s d o n e by E. Eldon a n d V. P i r r o t t a h a v e

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Figure 2. Summary of eve promoter elements and expression of the stripe 2 element. (A) The horizontal line represents a map of the eve promoter. Three essential cis regulatory elements were identified in earlier studies (Goto et al. 1989; Harding et al. 1989): an auto regulatory element located between - 5 . 9 and - 5 . 2 kb, the stripe 3 initiation element between - 3 . 8 and - 2 . 9 kb, and the stripe 2 and 7 elements located between - 1.7 and - 1.1 kb. The arrow and + 1 indicate the transcription initiation site. (B) Diagram of a heterologous fusion promoter that expresses stripe 2 in P-transformed embryos, eve 5' se- quences from - 2 . 9 kb to - 4 2 bp were inserted into the HZ50 P-element expression vector {Hiromi and Gehring 1987). HZ50 contains the hspTO min- imal promoter attached to the bacterial lacZ-coding sequence. (C) Embryo collected from a P-transformed line containing the fusion promoter shown in B. Expression of the lacZ reporter gene was detected by staining with an anti-~-galactosidase antibody. Staining is restricted to stripe 2; none of the other eve stripes are observed (including 7). (D) Stripe 2 expression ila a Kr- embryo. The eve--lacZ fusion gene shown in B was crossed into a Kr 9 (Rede- mann et al. 1988) mutant background. A broad band of lacZ expression is observed due to an expansion in the posterior limit of the stripe. (E) Stripe 2 expression in a g t - embryo. The eve-lacZ fusion gene shown in B was crossed into a gt YAs2 (Wieschaus et al. 1984) mutant background. A broader band of lacZ expression is observed due to expansion of the anterior border of the stripe.

s h o w n t h a t gt c o n t a i n s a basic l e u c i n e z i p p e r (pers. c o m m . ) . T w o sites of D N a s e I p r o t e c t i o n are observed, o n e l o c a t e d at a b o u t - 1 4 3 0 bp a n d a second at - 1 3 5 0 bp. T h e d i s t a l site c o m p l e t e l y e n c o m p a s s e s one of t h e five b e d - b i n d i n g sites p r e s e n t in t h e stripe 2 e l e m e n t . A m o n g t h e t h r e e g t - b i n d i n g sites t h a t w e h a v e identified, t h e o n e l o c a t e d at - 1350 bp c o n t a i n s t h e h i g h e s t a f f i n i t y for t h e p r o t e i n . I n t e r e s t i n g l y , t h e core s e q u e n c e w i t h i n t h e p r o t e c t e d r e g i o n p o s s e s s e s w e a k dyad s y m m e t r y a n d s h a r e s I 2 o u t of 20 i d e n t i t i e s w i t h t h e c o n s e n s u s se- q u e n c e of t h e p r o t o t y p i c l e u c i n e zipper p r o t e i n C/EBP (Fig. 3D; V i n s o n et al. 1989). T h e w e a k e r gtobinding sites at - 1430 a n d - 1110 bp s h a r e 10 o u t of 20 i d e n t i t i e s .

T h e l o c a t i o n s of bed-, hb-, gt-, a n d Kr-binding sites are s u m m a r i z e d in Figure 4. T h e r e are a t o t a l of 17 high- a f f i n i t y b i n d i n g sites b e t w e e n - 1700 a n d - 8 0 0 bp, a n d 12 of t h e s e m a p w i t h i n t h e 480obp region t h a t is e s s e n t i a l for e x p r e s s i o n (from - 1550 to - 1070 bp). As s h o w n pre- v i o u s l y (Stanojevic et al. i989), t h e r e are a t o t a l of t h r e e Kr sites a n d one h b site w i t h i n t h i s i n t e r v a l . T h e c u r r e n t s t u d y led to t h e i d e n t i f i c a t i o n of five b e d sites a n d t h r e e gt sites. O f t h e 12 b e d o hb-, gt-, a n d Kr-binding sites p r e s e n t w i t h i n t h i s region, 8 are f o u n d in t w o c l u s t e r s of - 5 0 bp e a c h (see Fig. 4B). E a c h of t h e s e c l u s t e r s i n c l u d e s b i n d i n g sites for t w o p u t a t i v e a c t i v a t o r s a n d t w o over- l a p p i n g r e p r e s s o r s (Fig. 4C). T h e p r o x i m a l c l u s t e r (at a b o u t - 1 . 1 kb) c o n t a i n s a h i g h - a f f i n i t y b i n d i n g site for

e a c h of t h e p u t a t i v e stripe 2 r e g u l a t o r s , w h e r e a s the di- s t a l c l u s t e r c o n t a i n s t w o b e d a c t i v a t i o n sites r a t h e r t h a n o n e b e d site a n d one h b site.

T h e tight l i n k a g e of a c t i v a t o r - a n d r e p r e s s o r - b i n d i n g sites suggests t h a t gt a n d Kr m i g h t define t h e stripe 2 borders t h r o u g h a c o m p e t i t i o n m e c h a n i s m . Figure 3 C s h o w s f o o t p r i n t a s s a y s w i t h t h e b e d a n d Kr proteins us- ing a D N A f r a g m e n t t h a t c o n t a i n s t w o copies of the 54- bp p r o x i m a l c l u s t e r (see s u m m a r y of s e q u e n c e in Fig. 4C). I n c r e a s i n g a m o u n t s of t h e b e d a n d Kr p r o t e i n s r e s u l t in sites of p r o t e c t i o n t h a t overlap e x t e n s i v e l y , a n d D N A - b i n d i n g studies w i t h m i x t u r e s of t h e t w o p r o t e i n s indi- c a t e t h a t t h e y c a n n o t c o - o c c u p y t h e s e closely l i n k e d sites (data n o t shown). T h i s close l i n k a g e probably re- s u l t s f r o m t h e s i m i l a r s e q u e n c e s r e c o g n i z e d by the b e d a n d Kr proteins; t h e i r core c o n s e n s u s - b i n d i n g sites share 5 o u t of 10 m a t c h e s (Driever a n d N f i s s l e i n - V o l h a r d 1989; Stanojevic et al. 1989; T r i e s m a n a n d D e s p l a n 1989).

b e d a n d hb f u n c t i o n m u l t i p l i c a t i v e l y to a c t i v a t e stripe 2 p r o m o t e r s e q u e n c e s in c o t r a n s f e c t i o n assays

T o d e t e r m i n e h o w t h e bed, hb, Kr, a n d gt p r o t e i n s m i g h t r e g u l a t e stripe 2 expression, t r a n s i e n t c o t r a n s f e c t i o n as- s a y s (Han et al. 1989) w e r e done by u s i n g r e p o r t e r plas- m i d s t h a t c o n t a i n s e q u e n c e s f r o m t h e s t r i p e 2 p r o m o t e r e l e m e n t . In t h e e x p e r i m e n t s d e s c r i b e d here, a single copy

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Transcriptional regulation of e v e

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g g

Figure 3. Footprint assays with eve stripe 2 sequences and bcd, Kr, and gt proteins. 32P-Labeled D N A fragments from the stripe 2 element were incubated with increasing amounts of full-length bcd (A), gt (B), or b c d + Kr (C) protein made in bacteria. P r o t e i n - D N A complexes were partially digested with DNase I and electrophoresed on polyacrylamide-urea gels. (A) A 406-bp BalI-DraIII D N A fragment (see Fig. 4A) from the stripe 2 element was 32P-labeled and incubated with affinity-purified b c d protein (lanes 2 and 3). The two regions that are protected by b c d are shown by the brackets to the right of the autoradiogram, along w i t h their sequences. Note the appearance of hypersensitive sites that flank the protected regions. Lanes labeled - correspond to DNA-binding reac-

tions with control extracts from bacteria lacking the b c d expression plasmid. Lane ga shows the G + A sequence of the D N A fragment using the Maxam-Gilbert sequencing reaction. (B) A 349-bp StyI-RsaI fragment was 32P-labeled and incubated with increasing amounts of bacterial extract containing full-length gt protein (lanes 2-5}. Two protected regions are shown by brackets, along with their sequences. (C) A 350-bp XbaI-KpnI D N A fragment containing two copies of the 54-bp proximal element was a2p-labeled at the XbaI site and incubated with increasing amounts of affinity-purified b c d protein (lanes 1 and 2) or Kr extract (lanes 4 and 5). Two protected areas (one in each copy of the 54-base element) were detected for each protein. The limits and sequence of the bcd-binding site is shown to the left of the autoradiogram, and the Kr site is shown to the right. N o t e that the protected regions overlap by at least 7 bp. (D) gt-binding sites are related to the C/EBP consensus sequence. Maximal alignments of the three gt-binding sites identified in this study. These are compared with the C/EBP consensus sequence of Landschulz et al. (t989). Sequence identities are indicated by the boxes. The numbers to the left indicate the locations of the binding sites relative to the transcription start site.

of e a c h c l u s t e r of b i n d i n g s i t e s (distal and p r o x i m a l ) w a s p l a c e d i n t a n d e m u p s t r e a m of t h e h s p 7 0 m i n i m a l pro- m o t e r a n d a t t a c h e d to t h e b a c t e r i a l c h l o r a m p h e n i c o l a c e t y h r a n s f e r a s e (CAT) reporter g e n e ( s u m m a r i z e d i n Fig. 5). S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h reporter plas-

m i d s c o n t a i n i n g e i t h e r t h e p r o x i m a l or d i s t a l c l u s t e r i n i s o l a t i o n (data n o t s h o w n ) . T h e reporter p l a s m i d s h o w n i n Figure 5 w a s u s e d to t r a n s f e c t S c h n e i d e r ceils, to- g e t h e r w i t h e x p r e s s i o n p l a s m i d s c o n t a i n i n g t h e full- l e n g t h b c d - , h b - , Kr-, and g t - c o d i n g s e q u e n c e u n d e r t h e

GENES & D E V E L O P M E N T 831

Cold Spring Harbor Laboratory Press on November 5, 2015 - Published by genesdev.cshlp.orgDownloaded from

S m a l l e t a l .

o ~ ~. =~ ~ +1 X rr~ ¢ v e-~ 03 f ~

II I I I - 1724

- 1 5 9 0 - 1 4 5 0 - 1 4 3 0

II 1-909

" - . . 100 bp

- 1 3 5 0 - 1 2 7 0 - 1 1 1 0 - 1 0 7 5 - 1 0 2 5 - 9 8 1 ~ ~ ~ ! l i ~ i t ~ ' ~ ~ v ' ~ ,

• • O O ® 0 - I 2 8 5 - 1 1 9 0 - 1 0 1 5 - 9 3 0

[ I 480 bp deletion

100 bp

C. proximal cluster (54 bp):

I hb ~ 1 I bcd t cgcgcattaggaag tcataaaaacacataataatg atg tcgaagggat tagggg

gtaatccttcag tatttttgtgtattattactacagcttccctaatccccgcgc I g t I I K r - - I

distal cluster (51 bp): I - - bcd -----7 j bcd ~ 1

cgtg ttaatccgtttgccatcagcgagattattag tcaattgcagttgcag caattaggcaaacggtagtcgctctaataatcagttaacgtcaacgtcgca

I K r I I g t I

Figure 4. Summary of the bcd-, hb-, Kr-, and gt-binding sites in the stripe 2 element. (A) Restriction map of the proximal region of the eve promoter. This is only a partial map showing some of the restriction sites relevant to the study. (B) Schematic map of the DNA-binding sites. The locations of the hb and Kr sites were reported previously (Stanojevic et al. 1989); the bcd and gt sites were identified in this study. The binding sites of activators (bcd and hb) are depicted as solid circles and stippled circles, respectively, below the line. Repressor sites (Kr and gt) are shown as hatched boxes and open boxes, respectively, above the line. Note the two clusters of binding sites (at - 1440 and - 1090), where two activator sites overlap two repressor sites. The 480-bp region (from the BssHII site to the BstEII site) that is essential for expression of stripe 2 (Goto et al. 1989) is shown below the line. (C) D N A sequence of the proximal and distal clusters showing the limits of the binding sites based on footprint assays. Oligonucleotides used for cotransfection assays correspond to the sequences shown here (see Fig. 5).

c o n t r o l of t h e a c t i n 5C p r o m o t e r (Driever a n d Nfisslein- V o l h a r d 1989). Each e x p r e s s i o n p l a s m i d w a s t e s t e d sep- arately; of t h e four, o n l y t h e bcd a n d hb p l a s m i d s acti- v a t e d C A T to a n y s i g n i f i c a n t e x t e n t . I n c r e a s i n g a m o u n t s of t h e actin-bcd e x p r e s s i o n p l a s m i d r e s u l t e d in a pro- gressive i n c r e a s e in C A T a c t i v i t y , w h i c h p e a k e d at a 17- to 18-fold i n d u c t i o n above b a c k g r o u n d levels (Table 1A). T h e h i g h e s t levels of t h e bcd e x p r e s s i o n p l a s m i d t h a t w e r e a s s a y e d r e s u l t e d in l o w e r i n c r e a s e s in C A T activ- ity, p o s s i b l y due to a s q u e l c h i n g effect (Gill a n d P t a s h n e 1988). T h e s e r e s u l t s suggest t h a t t h e bcd p r o t e i n binds to one or m o r e of t h e sites p r e s e n t in t h e s t r i p e 2 s e q u e n c e s a n d a c t i v a t e s t r a n s c r i p t i o n .

S i m i l a r e x p e r i m e n t s w e r e p e r f o r m e d w i t h a n expres- sion p l a s m i d ( p A c t S C - h b ) c o n t a i n i n g t h e f u l l - l e n g t h hb- coding s e q u e n c e . I n c r e a s i n g a m o u n t s of hb r e s u l t e d in o n l y m o d e s t i n c r e a s e s in C A T a c t i v i t y (Table 1B). H o w - ever, c o e x p r e s s i o n of t h e hb p l a s m i d a l o n g w i t h bcd re- s u l t e d in m u l t i p l i c a t i v e a c t i v a t i o n , w i t h as m u c h as a 44-fold s t i m u l a t i o n in C A T a c t i v i t y (Table 1C). A n i m -

p o r t a n t i m p l i c a t i o n of t h i s r e s u l t is t h a t t h e bcd m o r - p h o g e n m i g h t r e g u l a t e gene e x p r e s s i o n in e a r l y e m b r y o s by i n t e r a c t i n g w i t h t h e hb p r o t e i n (see D i s c u s s i o n l .

Repression by Kr and gt requires D N A binding

C o t r a n s f e c t i o n of Kr or gt e x p r e s s i o n p l a s m i d s e i t h e r a b o l i s h e d or m a r k e d l y r e d u c e d t h e m u l t i p l i c a t i v e acti- v a t i o n o b t a i n e d w i t h bcd a n d h b {Tables 2 a n d 3). In t h e s e e x p e r i m e n t s a m o u n t s of t h e bcd a n d hb e x p r e s s i o n p l a s m i d s w e r e u s e d t h a t r e s u l t e d in p e a k {44-foldl acti- v a t i o n of t h e r e p o r t e r p l a s m i d . C o t r a n s f e c t i o n s w i t h in- c r e a s i n g a m o u n t s of a p A c t 5 C - K r e x p r e s s i o n p l a s m i d c a u s e d as m u c h as a 22-fold r e d u c t i o n in t h e a c t i v a t i o n m e d i a t e d b y b c d + hb (Table 2A). A s i g n i f i c a n t reduc- t i o n in C A T a c t i v i t y w a s o b t a i n e d w i t h o n l y 0.2 txg of t h e Kr e x p r e s s i o n p l a s m i d , w h i c h is e q u i v a l e n t to t h e a m o u n t of hb a n d j u s t t w o f o l d m o r e t h a n t h e bcd re- q u i r e d for p e a k a c t i v a t i o n .

S i m i l a r c o t r a n s f e c t i o n e x p e r i m e n t s d o n e w i t h a

8 3 2 G E N E S & D E V E L O P M E N T

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Transcriptional regulation of ere

A °

- 1 5 9 0

B .

/ i

PT".~ I

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I I~)'A'ZI • • _! o o

- 9 3 0

:: - _ . . - -

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i -47 +~-~ +90 a~g SV40 poly A ,,,,).,,,,,,- ]] HH[[[]]H[H]H] H]]] [[H[]]H] [[ i!iiiiii!ilil [••••••!::i••••••i••••i!!•••!ii•••••i•!••!••i•i••••!i!i!!!!•!!!!;•••!!!•iii!!•••••i•;•••!•;!!••!•!•••!••!•i!••ii•i•i•iiiiiii;••i•ii•ii•iii•ii•••iiiii;i• i

16 bp HSP TATA CAT coding region

Figure 5. Schematic of CAT gene fusion construct used for cotransfection assays. The relationship of the binding sites within the stripe 2 element (A) to the CAT gene fusion construct (B) is shown. The reporter contains a 137-bp region of the Drosophila hsp70 gene, which includes 47 bp of the immediate 5'-flanking region and a 90-bp untranslated sequence. This m i n i m a l hsp70 promoter was attached to the bacterial CAT-coding sequence, which is flanked at its 3' end by SV40 polyadenylation sequences. A single copy of each cluster of binding sites was fused in tandem upstream of the minimal HSP70 promoter (see Materials and methods). These two clusters are separated by 16 bp of sequences from the polylinker.

p A c t 5 C - g t e x p r e s s i o n p l a s m i d suggest t h a t gt is a n e v e n m o r e effective repressor of stripe 2 e x p r e s s i o n t h a n Kr i x-~,.~ o,~1. . . x . u s e u. a n . n u . v . . . , . ~ ~ I . [ , L U U , t l L u ~ L.LI~., 6 ~ expression plasmid nearly abolished bcd-hb activation, r e d u c i n g C A T a c t i v i t y to t h e s a m e b a c k g r o u n d l e v e l as t h a t o b t a i n e d i n t h e a b s e n c e of e x p r e s s i o n p l a s m i d s .

Even t h o u g h Kr- a n d g t - b i n d i n g sites are c l o s e l y l i n k e d to b c d and h b a c t i v a t i o n sites w i t h i n t h e stripe 2 ele- m e n t (see s u m m a r y of s e q u e n c e i n Fig. 4B), t h e experi- m e n t s o u t l i n e d above do n o t r u l e o u t t h e p o s s i b i l i t y t h a t r e p r e s s i o n i n v o l v e s a n o n s p e c i f i c s q u e l c h i n g m e c h a n i s m (Levine a n d M a n l e y 1989). To e x c l u d e t h i s p o s s i b i l i t y we a n a l y z e d t h e a c t i v i t i e s of m u t a n t Kr a n d gt p r o t e i n s t h a t are u n a b l e to b i n d D N A .

A m u t a n t f o r m of t h e Kr p r o t e i n w a s s y n t h e s i z e d (see M a t e r i a l s a n d m e t h o d s ) t h a t m i m i c s t h e p r o d u c t en- coded b y a n u l l m u t a t i o n i n t h e gene, called Kr 9 (Rede- m a n n et al. 1988). Kr 9 is s t a b l y expressed i n h o m o z y - gotes a n d is i d e n t i c a l to t h e w i l d - t y p e p r o t e i n e x c e p t for a single a m i n o a c i d s u b s t i t u t i o n ; one of t h e h i g h l y con- served c y s t e i n e r e s i d u e s i n t h e s e c o n d z i n c finger is sub- s t i t u t e d w i t h a s e r i n e (Rosenberg et al. 1986; R e d e m a n n et al. 1988; Fig. 6A). Such a s u b s t i t u t i o n p r o b a b l y pre- v e n t s t h e b i n d i n g of a Z n 2 + atom, t h e r e b y d i s r u p t i n g t h e " f i n g e r " s t r u c t u r e a n d i m p a i r i n g its a b i l i t y to b i n d D N A (Rhodes a n d Klug 1988). G e l - s h i f t assays i n d i c a t e t h a t t h e m u t a n t Kr 9 p r o t e i n fails to b i n d to e i t h e r Kr site c o n t a i n e d i n t h e reporter p l a s m i d (data n o t shown). W h e n t e s t e d i n c o t r a n s f e c t i o n assays, t h e Kr 9 p r o t e i n w a s u n a b l e to repress t h e m u l t i p l i c a t i v e a c t i v a t i o n m e - diated b y t h e b c d a n d h b p r o t e i n s (Table 2B). E v e n t h e h i g h e s t l e v e l s of t h e Kr 9 e x p r e s s i o n p l a s m i d t h a t were assayed f a i l e d to reduce C A T expression. To a s s u r e t h a t t h i s l a c k of r e p r e s s i o n w a s n o t due to i n s t a b i l i t y of t h e m u t a n t protein, w e c o m p a r e d t h e e x p r e s s i o n of t h e Kr 9 p r o t e i n to w i l d - t y p e Kr i n p a r a l l e l c o t r a n s f e c t i o n exper- i m e n t s b y i m m u n o f l u o r e s c e n c e s t a i n i n g (Fig. 6B). T h e two p r o t e i n s are expressed at c o m p a r a b l e levels. More-

over, b o t h p r o t e i n s are r e s t r i c t e d to n u c l e i , i n d i c a t i n g t h a t t h e a m i n o a c i d s u b s t i t u t i o n i n t h e Kr 9 p r o t e i n does l~u~-"* ...o~n~ . . . . . ~e~ n o r m a l n u c l e a r transport.

In a s i m i l a r series of e x p e r i m e n t s a m u t a n t f o r m of t h e gt p r o t e i n (gt B.4) w a s s y n t h e s i z e d a n d tested, gt B.4 con- t a i n s 2 a m i n o a c i d c h a n g e s i n t h e b a s i c r e g i o n t h a t is a d j a c e n t to t h e l e u c i n e zipper p r e s e n t n e a r t h e c a r b o x y l t e r m i n u s (Fig. 6C). It h a s b e e n suggested t h a t t h i s b a s i c region s u p p l i e s t h e c o n t a c t p o i n t s n e c e s s a r y for D N A b i n d i n g v i a t h e " s c i s s o r s - g r i p " m o d e l (Vinson et al. 1989). W h e n p r o d u c e d i n a b a c t e r i a l e x p r e s s i o n s y s t e m , t h e m u t a n t gt p r o t e i n fails to b i n d to e i t h e r site i n t h e reporter p l a s m i d (data n o t shown). In t r a n s i e n t cotrans- f e c t i o n assays, t h e gt B.4 p r o t e i n also f a i l e d to repress C A T a c t i v a t i o n m e d i a t e d b y . b c d and h b (Table 3B). Im- m u n o f l u o r e s c e n c e e x p e r i m e n t s s h o w e d t h a t t h e gt B.4 p r o t e i n w a s s t a b l y e x p r e s s e d i n n u c l e i (Fig. 6D), exclud- i n g t h e p o s s i b i l i t y t h a t t h e f a i l u r e to repress w a s d u e to p r o b l e m s of s t a b i l i t y or i n t r a c e l l u l a r transport. T h i s ex- p e r i m e n t suggests t h a t direct D N A b i n d i n g is r e q u i r e d for g t - m e d i a t e d r e p r e s s i o n i n t h i s assay.

D i s c u s s i o n

We h a v e p r e s e n t e d a m o d e l for t h e i n i t i a t i o n of e v e stripe 2, w h e r e b y t h e m a t e r n a l m o r p h o g e n b c d a n d t h e gap p r o t e i n h b t o g e t h e r d e f i n e a broad a c t i v a t i o n d o m a i n i n t h e a n t e r i o r t h i r d of t h e e m b r y o . T h e borders of t h e stripe d e p e n d on s e l e c t i v e r e p r e s s i o n b y t h e gap p r o t e i n gt i n a n t e r i o r r e g i o n s a n d Kr p r o t e i n i n p o s t e r i o r regions. P r e v i o u s s t u d i e s h a v e e s t a b l i s h e d t h a t t h e n o r m a l s p a t i a l a n d t e m p o r a l l i m i t s of bcd, hb, Kr, and gt e x p r e s s i o n are c l o s e l y l i n k e d w i t h t h e stripe 2 p a t t e r n (Stanojevic et al. 1989; W a r r i o r a n d L e v i n e 1990; Kraut a n d Levine, 1991). Here, w e p r o v i d e a d d i t i o n a l e v i d e n c e t h a t t h e s e regula- tory factors d i r e c t l y r e g u l a t e stripe 2 e x p r e s s i o n , as s u m - m a r i z e d i n Figure 1D. First, t h e borders of a n e v e - I a c Z f u s i o n gene t h a t s e l e c t i v e l y e x p r e s s e s stripe 2 are ex-

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Small et al.

T a b l e 1. bcd and hb activate C A T via the proximal~distal clusters of binding sites

A. bcd alone B. hb alone

bcd (lag) CAT hb (p,g) CAT

0.0 1.0 0.0 1.0 0.01 5.2 0.01 1.6 0.025 10.4 0.025 2.6 0.05 12.0 0.05 3.0 0.1 16.9 0.1 3.8 0.2 16.1 0.2 4.4 0.4 17.6 0.4 4.5 0.8 12.8 0.8 4.2 1.6 12.6 1.6 2.0

C. bcd plus hb

bcd (lag) hb (lag) CAT

0 . 0 0 . 0 1.0 0.01 0.01 12.0 0.01 0.025 15.5 0.01 0.05 16.3 0.025 0.025 23.1 0.025 0.05 28.6 0.025 0.1 32.6 0.05 0.05 32.0 0.05 0.1 36.8 0.05 0.2 41.9 0.1 0.05 30.5 0.1 0.1 36.3 0.1 0.2 44.2

Transient cotransfection assays were performed as described in Materials and Methods. Increasing amounts of the pAct5C-bcd expression plasmid (A), the pActSC-hb plasmid (B), or both (C) were used along w i t h 1.0 lag of HSP-CAT reporter construct containing one copy each of the 51-bp distal element and one copy of the 54-bp proximal element in tandem (see Fig. 5). pActSC plasmid (without insert) was added where necessary so that the total a m o u n t of expression plasmid was the same in each transfection. The CAT activities shown are relative a m o u n t s compared to the baseline activity obtained w i t h the pAct5C plasmid alone (line 1 of A-C).

T a b l e 2. The C A T activation mediated by bcd and hb is repressed by wild-type Kr protein but not by Kr 9

A. Kr

bcd (lag) hb (txg) Kr (lxg) CAT

0.1 0.2 0 . 0 44.2 0.1 0.2 0.05 33.6 0.1 0.2 0.2 11.6 0.1 0.2 0.8 2.0

B. Kr 9

bcd (lag) hb (lag) Kr 9 (lag) CAT

0.1 0.2 0.0 44.2 0.1 0.2 0.05 45.8 0.1 0.2 0.2 53.1 0.1 0.2 0.8 47.8

Cotransfections were done w i t h 0.1 lag of pAct5C-bcd expres- sion plasmid and 0.2 I~g of p A c t 5 C - h b along w i t h increasing amounts of pAct5C-Kr (A) or pAct5C-Kr 9 (B) and 1.0 lag of the HSP-CAT reporter construct shown in Fig. 5.

t a t i o n h i e r a r c h y as a l i n e a r s e r i e s of g e n e i n t e r a c t i o n s . H o w e v e r , a l t h o u g h o u r r e s u l t s a r e c o n s i s t e n t w i t h a di- r e c t r o l e for bcd, i t is c o n c e i v a b l e t h a t b c d a c t s i n d i r e c t l y o n s t r i p e 2 e x p r e s s i o n v i a h b . It s h o u l d b e n o t e d t h a t t h e r e is a p r e c e d e n t for t h e d i r e c t i n v o l v e m e n t of a m a - t e r n a l f a c t o r w i t h a p a i r - r u l e p r o m o t e r : T h e s o - c a l l e d ze- b r a e l e m e n t of t h e f u s h i t a r a z u (ftz) p r o m o t e r ( H i r o m i e t al. 1985) a p p e a r s t o be d i r e c t l y a c t i v a t e d b y t h e h o m e o b o x p r o t e i n c a u d a l (cad) ( D e a r o l f e t al. 1989).

T h e d e m o n s t r a t i o n t h a t c o m b i n a t i o n s of t h e b c d a n d h b p r o t e i n s c a n m u l t i p l i c a t i v e l y a c t i v a t e a r e p o r t e r g e n e c o n t a i n i n g s t r i p e 2 s e q u e n c e s s u g g e s t s t h a t b c d m i g h t n o t i n f l u e n c e t h e s e g m e n t a t i o n p a t t e r n s o l e l y t h r o u g h t h e r e g u l a t i o n of gap genes. P r e v i o u s s t u d i e s h a v e s h o w n t h a t t h e b c d p r o t e i n is d i s t r i b u t e d i n a b r o a d c o n c e n t r a -

p a n d e d w h e n c r o s s e d i n t o g t - or K r - e m b r y o s . M o r e - over, D N A - b i n d i n g e x p e r i m e n t s i n d i c a t e t h a t bcd, hb, Kr, a n d gt p r o t e i n s b i n d w i t h h i g h a f f i n i t y t o s e q u e n c e s c o n t a i n e d w i t h i n t h e s t r i p e 2 p r o m o t e r e l e m e n t . F i n a l l y , w e h a v e s h o w n t h a t - 1 0 0 bp of s t r i p e 2 p r o m o t e r se- q u e n c e s m e d i a t e a c t i v a t i o n b y b c d a n d h b p r o t e i n s a n d r e p r e s s i o n b y gt a n d Kr.

D o e s b c d d i r e c t l y r e g u l a t e e v e expression?

T h e i d e n t i f i c a t i o n of m u l t i p l e , h i g h - a f f i n i t y b c d - b i n d i n g s i t e s i n t h e s t r i p e 2 e l e m e n t s u g g e s t s t h a t t h e b c d m o r - p h o g e n c o u l d p l a y a d i r e c t r o l e i n a c t i v a t i n g e v e expres- s i o n i n e a r l y e m b r y o s . T h r e e of t h e s e b i n d i n g s i t e s m e - d i a t e s t r o n g a c t i v a t i o n b y t h e b c d p r o t e i n i n c o t r a n s f e c - t i o n a s s a y s . T h e p o s s i b i l i t y t h a t b c d d i r e c t l y r e g u l a t e s e v e c h a l l e n g e s t h e s t r i c t e s t i n t e r p r e t a t i o n of t h e s e g m e n -

T a b l e 3. The C A T activation mediated by bcd and hb is repressed by wild-type gt protein but not by gt B.4

A. gt

bcd (lag) hb (lag) gt (lag) CAT

0.1 0.2 0.0 44.2 0.1 0.2 0.05 9.1 0.1 0.2 0.2 1.2 0.1 0.2 0.8 0.7

B. gtB.4

bcd (lag) hb (lag) gt B.4 (lag) CAT

0.1 0.2 0.0 44.2 0.1 0.2 0.05 42.3 0.1 0.2 0.2 43.9 0.1 0.2 0.8 40.2

Cotransfections were done as in Table 2, except w i t h increasing amounts of pAct5C-gt (A) or p A c t 5 C - g t B.4 {BI.

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Transcriptional regulation of e v e

a. C. / R - - D'-- H / R - - D-_ H

/T \ / T \ F H H / ~ F \

L I L R K r W r I R K r 9 I I K I K K / K

D T \ / / D T /

(-~ 255 H ~ M (~)255 H . . M ,~\ / ~ ~

++ R I R p Zn / \ . - ' / ..~C H / L/

E N 250 / T 273 250 / E \ T 273

F F

B, D.

NH COOH C ( ~ H NH 2 2

-L L-

oll "'

m E R R R K N N A A A K K S R D R R R I K E D E gt B'4 ERRRKNNAAAKKS RDR~ I KED E

Figure 6. S u m m a r y of Kr and g t p r o t e i n s u s e d i n c o t r a n s f e c t i o n assays. (A) P r e d i c t e d s t r u c t u r e of t h e s e c o n d of four Z n 2+ fingers i n t h e w i l d - t y p e {left) a n d Kr 9 m u t a n t (right) p r o t e i n s ( R e d e m a n n et al. 1988). A m i n o acid r e s i d u e s are r e p r e s e n t e d by t h e s i n g l e - l e t t e r _ _ _1 _ r ~ - _ _ Q . . . . _. . . . . . . . _. J . . . . . . . . . . . . . ] coue. r,: c o n t a i n s a 8¢Hi1¢ t¢~iuu¢ at p o s i t i o n ,oo " . . . . . , 1 p,a~c' . . . . v,r o n e of "~t,,c- ,,~,,,y~-: _LI.. L.u11~c, you cysteir, e r e s i d u e s i n t h e w i l d - t y p e p r o t e i n . T h i s s u b s t i t u t i o n s h o u l d p r e v e n t t h e f o r m a t i o n of a f u n c t i o n a l Z n z+ finger and i m p a i r s its a b i l i t y to b i n d D N A (data n o t shown). (B) Expression of t h e w i l d - t y p e (left) a n d Kr 9 (right) p r o t e i n s i n t r a n s f e c t e d S c h n e i d e r cells. Both p r o t e i n s are stably expressed i n n u c l e i . P r o t e i n s w e r e d e t e c t e d w i t h a n t i - K r a n t i b o d i e s a n d v i s u a l i z e d by i n d i r e c t i m m u n o f l u o r e s c e n c e . (C) P r e d i c t e d s t r u c t u r e of a g t p r o t e i n d i m e r by a s s o c i a t i o n of p u t a t i v e l e u c i n e zippers {after L a n d s c h u l z et al. 1989). T h e s e q u e n c e of t h e basic r e g i o n a d j a c e n t to t h e l e u c i n e zipper is s h o w n for t h e w i l d - t y p e g t p r o t e i n and t h e s y n t h e t i c m u t a n t gt B.4. (D) E x p r e s s i o n of t h e w i l d - t y p e (left) and t h e gtB.4 (right) p r o t e i n s i n t r a n s f e c t e d S c h n e i d e r cells. Both p r o t e i n s are stably expressed in n u c l e i .

tion gradient w i t h peak levels at the anterior pole (Driever and Nfisslein-Volhard 1988). O n l y levels of bed protein above a m i n i m a l threshold can activate hb, re- sulting in a relatively sharp border of h b expression in anterior regions (Driever et al. 1989; Struhl et al. 1989). This threshold process could, in principle, generate two distinct c o m b i n a t i o n s of regulatory activity, w i t h em- bryonic cells in anterior regions containing both the bed and h b proteins while more posterior cells express only bed. Several types of m e c h a n i s m s could account for mul- tiplicative interactions between the bed and h b proteins, including cooperative binding to D N A or so-called " p r o m i s c u o u s " cooperativity involving p r o t e i n - p r o t e i n i n t e r a c t i o n s w i t h the transcription m a c h i n e r y (Lin et al. 1990).

M e c h a n i s m of repression

Genetic studies have shown t h a t relatively small changes in the level of Kr protein cause a significant expansion in the l i m i t s of eve stripe 2 expression (Frasch and Levine 1987; Warrior and Levine 1990). Thus, the broad, bell-shaped distribution profile of Kr expression in central regions of t h e e m b r y o dictates a relatively sharp on/off s w i t c h in eve expression (see Fig. 1D). Although the Kr expression p a t t e r n has n o t been quantified, it

would appear t h a t on the order of a twofold change in the level of repressor is sufficient to trigger this switch. Such threshold repression m i g h t be a m a n i f e s t a t i o n of coop- erativity between activators. Three of the five bccl-bind- ing sites present w i t h i n t h e stripe 2 e l e m e n t are closely linked to a Kr site. In principle, Kr m i g h t repress expres- sion by competing w i t h t h e binding of bed to just one or two of the sites, as this w o u l d disrupt potential cooper- ative interactions among t h e activators.

A related m e c h a n i s m of repression is t h a t t h e binding of gt or Kr to a given site interferes w i t h the activity, but not the binding, of neighboring bed proteins. For exam- ple, gt bound to the proximal cluster should block the binding of the h b activator (see Fig. 4B). Perhaps gt also represses by m a s k i n g or " q u e n c h i n g " the a c t i v i t y of bed bound at the neighboring site (Levine and M a n l e y 1989).

T r a n s i e n t cotransfection assays suggest t h a t gt is a more effective repressor t h a n Kr (see Tables 2 and 3). T h e greater effectiveness of gt reflects the s i t u a t i o n in the embryo, in t h a t gt is active in regions where there are higher levels of the bed and h b activators (see Fig. 1D). In contrast, as discussed above, Kr is active in regions con- taining only low levels of these activators. It should be noted, however, t h a t repression by gt is n o t sufficient to a c c o u n t for the e s t a b l i s h m e n t of the stripe 2 border in vivo. There is a t r a n s i e n t and i n c o m p l e t e fusion of

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S m a l l e t al.

s t r i p e s 1 a n d 2 i n g t - e m b r y o s (Frasch a n d L e v i n e 1987), r a i s i n g t h e p o s s i b i l i t y t h a t a d d i t i o n a l repressors operate to k e e p t h e stripe 2 e l e m e n t off i n a n t e r i o r regions con- t a i n i n g h i g h l e v e l s of t h e b c d a n d h b activators.

Short-range repression p e r m i t s a u t o n o m o u s a c t i o n of stripe e l e m e n t s

T h e e v e p r o m o t e r is c o m p o s e d of a series of separate cis s e q u e n c e s t h a t r e g u l a t e i n d i v i d u a l stripes, a l t h o u g h all of t h e s e e l e m e n t s act on a c o m m o n promoter. H o w do t h e s e e l e m e n t s act i n d e p e n d e n t l y of one another? A s i m - i l a r s i t u a t i o n h a s b e e n s h o w n for t h e h a i r y promoter, w h i c h is a n o t h e r p r i m a r y pair-rule gene c o n t a i n i n g sep- arate stripe i n i t i a t i o n e l e m e n t s (Howard et al. 1988; H o w a r d a n d S t r u h l 1990; P a n k r a t z et al. 1990).

T h e s u m m a r y figure s h o w n i n Figure 7 p r e s e n t s a m o d e l t h a t d e s c r i b e s h o w t w o stripe i n i t i a t i o n e l e m e n t s , 2 a n d 3, m i g h t f u n c t i o n a u t o n o m o u s l y . G e n e t i c s t u d i e s on t h e r e g u l a t i o n of stripe 3 are i n c o m p l e t e , b u t a rea- s o n a b l e p o s s i b i l i t y is t h a t h b (and p e r h a p s bcd) b o t h ac- t i v a t e s a n d represses i t s e x p r e s s i o n (Frasch a n d L e v i n e 1987; R. W a r r i o r a n d M. Levine, unpubl.). Approxi- m a t e l y 20 h b - b i n d i n g sites h a v e b e e n i d e n t i f i e d w i t h i n t h e stripe 3 e l e m e n t , w h i c h m i g h t p e r m i t its a c t i v a t i o n i n regions of t h e e m b r y o w h e r e t h e r e are l o w l e v e l s of t h e h b p r o t e i n (Stanojevic et al. 1989). A c t i v a t i o n m i g h t oc- c u r w h e n a s m a l l n u m b e r of h i g h - a f f i n i t y h b - b i n d i n g sites are filled. In m o r e a n t e r i o r regions t h e r e are h i g h l e v e l s of h b , w h i c h m i g h t r e s u l t i n b i n d i n g to m o s t or all of t h e sites, i n c l u d i n g l o w - a f f i n i t y sites t h a t m e d i a t e re- pression. T r a n s i e n t c o t r a n s f e c t i o n assays are c o n s i s t e n t w i t h t h i s m o d e l a n d h a v e s h o w n t h a t h b can a c t i v a t e or repress gene e x p r e s s i o n i n a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Zuo et al. 1991). T h e stripe 3 e l e m e n t m i g h t

evade r e p r e s s i o n b y e v e n h i g h c o n c e n t r a t i o n s of the Kr repressor, as it c o m p l e t e l y l a c k s h i g h - a f f i n i t y Kr-binding s i t e s (Stanojevic et al. 1989). In regions w h e r e stripe 3 is expressed t h e stripe 2 e l e m e n t is i n a c t i v e due to t h e b i n d i n g of Kr p r o t e i n to t h e three h i g h - a f f i n i t y sites it c o n t a i n s .

A n i m p o r t a n t i m p l i c a t i o n of t h i s m o d e l is t h a t t h e b i n d i n g of Kr to t h e stripe 2 e l e m e n t does n o t interfere w i t h t h e a c t i v i t y of t h e stripe 3 e l e m e n t , w h i c h is lo- cated - 1 . 5 kb a w a y (Goto et al. 1989; H a r d i n g et al. 1989; see s u m m a r y i n Fig. 2A). T h e i n a b i l i t y of Kr to repress over s u c h a d i s t a n c e is c o m p a t i b l e w i t h t h e c o t r a n s f e c t i o n studies, w h i c h suggest t h a t short-range r e p r e s s i o n is s u f f i c i e n t to a c c o u n t for t h e i n a c t i v a t i o n of stripe 2 expression. It is s t r i k i n g t h a t n e a r l y every one of t h e bcd- a n d h b - b i n d i n g sites p r e s e n t w i t h i n t h e stripe 2 e l e m e n t overlaps or is i m m e d i a t e l y a d j a c e n t to a gt or Kr repressor site. If long-range r e p r e s s i o n w e r e a n i m p o r t a n t m e c h a n i s m g o v e r n i n g e v e expression, t h e r e w o u l d be no n e e d to h a v e s u c h t i g h t l i n k a g e of t h e activators and repressors. S i m i l a r a r r a n g e m e n t s of a c t i v a t o r s a n d re- pressors h a v e b e e n i d e n t i f i e d i n n u m e r o u s m a m m a l i a n promoters, w h i c h suggests t h a t short-range repression m i g h t be i m p o r t a n t i n t h e s e s y s t e m s as w e l l (Maniatis et al. 1987). Short-range r e p r e s s i o n m i g h t a c c o u n t for t h e e v o l u t i o n of c o m p l e x p r o m o t e r s t h a t are c o m p o s e d of m u l t i p l e , a u t o n o m o u s r e g u l a t o r y e l e m e n t s .

Materials and m e t h o d s R e c o m b i n a n t p l a s m i d s

T h e eve stripe 2 e l e m e n t / h e a t s h o c k p r o m o t e r (HSP)/CAT re- p o r t e r p l a s m i d {Fig. 5) w a s c o n s t r u c t e d f r o m s y n t h e t i c oligonu- cleotides (sequences are s h o w n i n Fig. 4C). Each oligonucleotide set was annealed together, blunt-ended with Klenow, and

Figure 7. A model for the autonomous action of the stripe 2 and 3 elements. The curves repre- sent, only very approximately, the limits and lev- els of bcd, fib and Kr expression in the region of the embryo where eve stripes 2 and 3 are ex- pressed. The dashed vertical lines correspond to the stripe 2 and 3 borders. The region of stripe 2 expression contains relatively high levels of bcd and hb and low levels of Kr. In contrast, the re- gion of stripe 3 expression contains low levels of bcd and fib expression and very high levels of Kr. The horizontal lines on the bottom represent the activities of the eve promoter in regions of the embryo where stripes 2 and 3 are expressed. In the stripe 2 region many bcd- and fib-binding sites in the eve promoter are occupied (lower left), resulting in the activation of the stripe 2 element and the repression of the 3 element. Re- pression of the stripe 3 element might result from the binding of hb (and/or bcd) to low-affin- ity sites that mediate repression (see Discussion). In the stripe 3 region the high concentrations of

hb K r

b c d

e v e stripe 2 eve stripe 3

1#3 element I I #2 element I ~ 1#3 element ] I #2 element I hb hb hb bcd hb hb hb Kr Kr Kr

OFF ON ON OFF Kr repress the stripe 2 element, but not the stripe 3 element, because it lacks Kr-binding sites (Stanojevic et al. 1989). The lower levels of fib (and perhaps bcd) in this region might activate the stripe 3 element by binding only to high-affinity sites (see Discussion).

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Transcriptional regulation of e v e

cloned separately into the EcoRV site of pBluescript S K - (pur- chased from Strategene, La Jolla, CA) to generate intermediate clones pBS51 and pBS54. The 51-bp distal e l e m e n t was cloned upstream of the 54-bp proximal e l e m e n t by cutting pBS51 with EcoRI, blunt-ending, and cutting with KpnI to give a 90-bp frag- ment, w h i c h was ligated into pBS54 that was cut with HindIII, blunt-ended, and cut w i t h KpnI. This clone (pBS51/54) was se- quenced to verify that it contained both e l e m e n t s separated by 16 bases of Bluescript polylinker, pBS51/54 was then cut with EcoRI, blunt-ended, cut with HindIII, and inserted into HSP- CAT parental vector that had been cut w i t h SalI, blunt-ended, and cut w i t h HindIII. The promoter sequences from the stripe 2 region were also cloned into another CAT vector that contains the m e t a l l o t h i o n i n e m i n i m a l promoter in place of the HSP70 sequences (Han et al. 1989). In all cases, the two different basal promoters gave very similar results.

The bcd expression vector p A c t 5 C - b c d was prepared by clon- ing the N d e I - B a m H I fragment containing the bcd-coding re- gion from pAR-bcdNB (Driever and Niisslein-Volhard 1989) into pPacU + NDE (Biggin and Tjian 1989). This bcd protein contains two extra residues at the amino terminus (methionine and histidine). All other expression vectors were derived from the parental plasmid pActSCSRS, kindly provided by Dr. K. Bur- gess. This plasmid contains - 2 . 5 kb of 5'-flanking sequences from the Drosophila actin 5C gene. pAct5C-gt was constructed from a full-length gt cDNA (described by Kraut and Levine 1991). p A c t 5 C - h b and pAct5C-Kr were kindly provided by K. Hart {see Zuo et al. 1991). To make the p A c t 5 C - K r 9 expression plasmid, a -1500-bp NotI-KpnI fragment from the Kr-coding sequence was cloned into the polylinker of pBtuescript S K - . The Kr 9 m u t a t i o n was generated by oligonucleotide-directed mutagenesis by using the 31-nucleotide oligomer, 5'-GAA- TGTCCGGGGAAAGTGACAAGCGGTTTAC-3', which con- tains three mismatches w i t h the wild-type Kr sequence. These substitutions change a cysteine residue to a serine (according to the sequence of the Kr 9 m u t a n t reported by Redemann et al. 1988). Mutagenic clones were screened by sequence analysis. The -1500-bp NotI-KpnI-mutagenized fragment was t h e n sub- stituted back into the parent vector pActSC-Kr, verified by re- striction mapping and sequence analysis. The gt B.4 mutation was generated by oligonucleotide-directed mutagenesis by us- ing the 30-nucleotide oligomer 5'-GAACTACGCCGGCAGTA- GCGCCGTGAAGAA-3', w h i c h contains three mismatches w i t h the wild-type sequence (R. Kraut, unpubl.). The 1.4-kb NdeI-SacI fragment containing the mutagenized sequence was t h e n substituted back into the parental vector pAct5C-gt and verified by restriction and sequence analysis.

DNase I protection assays

Footprint assays were performed exactly as described by Hoey and Levine (1988). Protein extracts used for the binding assays w i t h Kr and gt were prepared w i t h guanidine HC1, followed by extensive dialysis, exactly as described by Hoey et al. (1988). The bcd protein used in the DNA-binding studies was affinity purified from the soluble fraction of the bacterial lysate essen- tially as described (Kadonaga and Tjian 1986) by using m u l t i m - ers of t h e A3 bcd-binding site from the h b promoter {Driever and Nfisslein-Volhard 1989).

A n t i b o d y staining of embryos

P transformants were stained w i t h a rabbit anti-B-galactosidase and detected by a histochemical procedure employing the ABC Elite k i t (purchased from Vector Labs, Burlingame, CA), exactly as described by the manufacturer. The stained embryos were

photographed, and the color transparencies were used to print the inverse image, as described by Harding et al. (1989). Immu- nolocalization of eve was done w i t h a rabbit anti-eve antibody, kindly provided by Dr. Manfred Frasch. gt was detected with a guinea pig antibody described by Kraut and Levine {1991), Kr was localized with a rabbit anti-Kr antibody kindly provided by Dr. Christine Rushlow, and h b was detected w i t h a mouse an- tibody kindly provided by David Kosman. Double immunoflu- orescence staining was done exactly as described by Stanojevic et al. (1989).

Cotransfections and transient expression assays

Drosophila Schneider $2M3 cells were grown in M3 m e d i u m (GIBCO) supplemented w i t h 10% defined fetal bovine serum {GIBCO), w h i c h was heat inactivated at 60°C for 30 rain. Cotransfections were performed essentially as reported in Han et al. (1989). For each transfection - 4 x 1 0 6 cells were plated per 60-mm tissue culture dish 1 day before transfection. In all cases, 1.0 ~g of a given eve element/HSP/CAT reporter plasmid was transfected along with various a m o u n t s of expression plas- mids and 2.0 ~g of Copia long-terminal repeat (LTR)-IacZ plas- mid, w h i c h served as an internal control for transfection effi- ciency (Han et al. 1989). pActSC plasmid vector (without in- serted coding sequences) was added where required to standardize the a m o u n t of expression plasmids in each experi- ment. The total a m o u n t of D N A in each transfection was ad- justed to 10 ~g by the addition of pGEM-1 or pUG18 as carrier. All experiments were performed at least twice. Preparations of cell extracts, fS-galactosidase assays, and CAT assays were de- scribed previously {Han et al. 1989). Indirect immunofluores- cence assays were performed as described by Rushlow et al. (1989). For the Kr experiments a rabbit anti-Kr primary antibody and a TRITC-conjugated anti-rabbit secondary antibody were used to detect Kr protein. For the gt experiments a guinea pig anti-gt antibody and a TRITC-conjugated anti-guinea pig sec- ondary antibody were used to detect gt protein.

A c k n o w l e d g m e n t s

We t h a n k Dr. Wolfgang Driever for the p A R b c d N B expression plasmid and Peter Lawrence for help w i t h rewriting the manu- script. We are grateful to K. Han and J. Colgan for assistance with the cotransfection assays. We also t h a n k Denise Boyd for technical assistance. We are grateful to Robert Tjian for permit- ting T.H. to perform the footprint assays in his laboratory. This work was funded by a grant from t h e National Institutes of Health (GM 34431 ).

The publication costs of this article were defrayed in part by payment of page charges. This article m u s t therefore be hereby marked " a d v e r t i s e m e n t " in accordance w i t h t8 USC section 1734 solely to indicate this fact.

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