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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9309-9314, September 1996 Colloquium Paper

This paper was presented at a colloquium entitled "Biology of Developmental Control, " organized by Eric H. Davidson, Roy J. Britten, and Gary Felsenfeld, held October 26-28, 1995, at the National Academy of Sciences in Irvine, CA.

Long-range repression in the Drosophila embryo HAINI N. CAI, DAVID N. ARNOSTI, AND MICHAEL LEVINE* Department of Biology, Center for Molecular , Pacific Hall, University of California at San Diego, La Jolla, CA 92093-0347

ABSTRACT Transcriptional can be character- Short-range transcriptional repression appears to account ized by their range of action on promoters and enhancers. for autonomy in a modular . Repressors Short-range repressors interact over distances of50-150 bp to bound to a given enhancer do not interfere with the activators inhibit, or quench, either upstream activators or the basal contained within neighboring enhancers. For example, the transcription complex. In contrast, long-range repressors act posterior border of eve stripe 3 is established by the gap over several kilobases to silence basal promoters. We describe knirps (kni; ref. 7), which is a member of the nuclear recent progress in characterizing the functional properties of receptor superfamily, and is expressed in the presumptive one such long-range element in the Drosophila embryo and abdomen in early embryos (8). There are at least five kni- discuss the contrasting types of regulation that are made binding sites in the stripe 3 enhancer, two of which map within possible by short- and long-range repressors. 100 bp of critical d-STAT sites within the enhancer (9). The range of kni repression activity was investigated by Complex patterns of in the Drosophila embryo inserting synthetic kni-binding sites into a well-defined heter- are specified by spatially localized activators and repressors. ologous enhancer, the rhomboid lateral stripe enhancer ("rho Mechanisms of transcriptional activation are beginning to be NEE"). The rho NEE is repressed in abdominal regions of elucidated, but repression mechanisms are still poorly under- transgenic embryos when kni-binding sites are placed within stood. A combination of genetic, molecular, and biochemical 50-100 bp of the activator sites. However, when the kni sites approaches have begun to address this issue. Several types of are separated by 150 bp from the nearest NEE activators, they transcriptional repression have been proposed (1, 2), including are ineffective in mediating repression (10). These and other direct competition of repressors and activators for common observations prompted the proposal that kni is a short-range binding sites to DNA, local "quenching" of upstream activa- repressor that functions in a local fashion to inhibit, or quench, tors or the basal transcription complex, and long-range inter- nearby activators within the enhancer to which it is bound. In actions between repressors and the basal apparatus. The principle, this type of repression can permit enhancer auton- emerging picture is that some factors mediate "short-range" omy, as summarized in Fig. 1 Upper. A synthetic modular repression, interfering with transcriptional activators bound promoter containing the eve stripe 3 enhancer and rho NEE within 50-150 bp, whereas other "long-range" repressors are directs an additive pattern of expression because rho NEE capable of interfering with promoter function over long dis- activators are located beyond the range of kni repressors tances (see Fig. 1). As we discuss below, one such long-range bound to the stripe 3 enhancer (10). repressor complex, the ventral repression element (VRE) Similar experiments suggest that other spatially localized from the zerknullt (zen) gene, appears to silence the transcrip- repressors function only over short distances and permit tion complex over a distance of several kilobases. enhancer autonomy in a modular promoter. Examples include Drosophila promoters are often modular and contain a series Kruppel, which defines the posterior border ofeve stripe 2, and of nonoverlapping enhancers, which function independently of snail, which is responsible for excluding rho NEE expression one another to of from the ventral (11, 12). Both Kruppel and snail generate composite patterns gene expres- to function over distances to kni. All sion. The gene, even-skipped (eve) contains a appear comparable three modular promoter. It encodes a repressors must map within -100-150 bp from the activators homeodomain that is that they quench. This requirement for close spacing ensures essential for the subdivision of the embryo into a repeating enhancer because enhancers are series of eve autonomy neighboring gen- body segments (3, 4). is transcribed in a series of erally beyond the range of repression. It is possible to "force" seven transverse stripes along the length of precellular em- a short-range repressor to block two enhancers. For example, bryos, foreshadowing overt segmentation of the germ band by as discussed above, the eve stripe 2 and stripe 3 enhancers are several hours (5). The expression of at least some of the stripes normally separated by a 1.7-kb spacer sequence. When this are regulated by separate enhancers in the eve promoter spacer is removed, the proximal-most Kruippel-binding site region. For example, stripe 3 is regulated by a 500-bp enhancer within the stripe 2 enhancer now maps within 150 bp of stripe located -3 kb upstream of the eve transcription start site (6). 3 activators. As a result, Kruppel not only represses the In contrast, stripe 2 is regulated by a separate 500-bp enhancer posterior border of stripe 2, but also attenuates the expression located - 1 kb from the start site. The stripe 2 and stripe 3 of stripe 3 (13). enhancers are separated by a 1.7-kb "spacer" sequence in the In contrast with the short-range repression discussed above, eve promoter region. A major goal of our recent studies has long-range repression works in a dominant fashion over dis- been to determine how these two enhancers function inde- tances of several kilobases. Such repression has been observed pendently of one another to generate a multistripe pattern of in a number of systems. For example, a long-range repression expression in early embryos. Abbreviations: VRE, ventral repression element; MSE, minimal stripe The publication costs of this article were defrayed in part by page charge enhancer; kni, knirp. payment. This article must therefore be hereby marked "advertisement" inI *To whom reprint requests should be addressed. e-mail: levine@ accordance with 18 U.S.C. §1734 solely to indicate this fact. jeeves.ucsd.edu. 9309 Downloaded by guest on September 29, 2021 9310 Colloquium Paper: Cai et al. Proc. Natl. Acad. Sci. USA 93 (1996) Short-range repression A

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Long-range repression VRE MSE r h\\\\\\\\\\l LI I-- eve/lacZ 4D~ 11 B FIG. 1. Short- and long-range repression. (Upper) A short-range repressor (circle with spiral) locally represses activators (ovals) on enhancer 1, inhibiting this element's activity. Activators in enhancer 2 are not affected, allowing enhancer 2 to activate the gene. (Lower) A long-range repressor (circle with spiral) bound to enhancer 1 domi- nantly represses the transcription complex and blocks both enhancers. element establishes the rhombomere-specific pattern of MSE -. VRE within hindbrain of mouse Hoxb-1 expression the the embryo taiaii I'.rxxxxxxxxxxx.- % \\\ (14). Here we present evidence for a Drosophila long-range eve/ I acZ repressor, which appears to be distinct from short-range repressors such as snail and Kriippel. The promoter region of C the zen gene directs a simple on/offpattern of expression in the early embryo. zen is activated by one or more ubiquitously distributed factors and is repressed in ventral and lateral regions by the rel-related , dorsal (dl). In a number of contexts, such as the rho NEE, dl acts as a transcriptional activator. However, in the context of the zen promoter, dl functions as a repressor. Previous studies have shown that dl binds near one or more "" sites within a distal region of the zen promoter (15-17). This distal sequence, the VRE, is located between -1.6 and -1.0 kb MSE VRE upstream of the zen transcription start site (Fig. 2). It contains three high-affinity dl-binding sites, as well as closely linked eve/latZ corepressor sites. Several lines of evidence suggest that the zen VRE functions FIG. 3. The zen VRE represses a heterologous enhancer over a as a dominant the exact of long distance. were detected by whole-mount in situ hybridiza- repressor element, although range tion, using a digoxigenin-labeled lacZ antisense RNA probe (6). repression was not established (15-17). The 600-bp VRE can Drosophila embryos were transformed by P-element-mediated germ- line transformation as described in ref. 6. Embryos are oriented with ZEN VENTRAL REPRESSION ELEMENT anterior to the left and dorsal up. (A-C) Early nuclear cycle 14 embryos containing VRE 600-MSE eve lacZ fusion . (A) zen VRE 600 is placed 5' of the . Early expression includes a N D1 D2 D3 XPn broad stripe 2 band in the anterior that is ventrally repressed by the oA2 A3 -1 .6 -1.0 i VRE 600, superimposed on the dorsal staining-mediated by general AT1 AT2 AT3 activator sequences present in the zen VRE (16, 18). MSE, minimal stripe enhancer. (B and C) zen VRE 600 placed downstream of the lacZ in the forward (B) and reverse (C) orientation. The VRE600 pattern of ventral repression is comparable to that of A. repress the ventral and lateral expression of various heterol- VRE200 ogous promoters and enhancers. For example, when placed immediately upstream of the eve stripe 2 enhancer, the VRE FIG. 2. The zen promoter region. The top horizontal bar represents transiently represses stripe 2 in ventral and lateral regions of the zen promoter region and the arrow corresponds to the transcription precellular embryos (16, 17). In these experiments, the dl- and start site. Open circles above the bar represent dl-binding sites in the corepressor-binding sites within the VRE map more than zen VRE (marked D1-D3). AT-rich corepressor sites 1-3 are indicated with solid below the bar. The VRE 600 and VRE 200 200-300 bp from the nearest stripe 2 activator sites. It is squares promoter conceivable that the bound to the elements are indicated by the horizontal bars below. Restriction "dl-corepressor complexes" sites used to isolate the fragments are indicated above the VRE might work in a local fashion to quench nearby activators, promoter. X, XbaI; N, NarI; Xmn, XmnI; H, HindIII. General acti- over somewhat longer distances (300 bp rather than 100-150 vators bind to the element 5' of the NarI site. bp seen for kni, Kriippel, and snail). Downloaded by guest on September 29, 2021 Colloquium Paper: Cai et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9311 A

1 2 3 MSE r 1 2 3 MSE x2 EE Z (..p3m3.inL) xx212 Iu [X- * ) eve/lacZ E30 ~~~~eve/lacZ B D _ , ,6 ... ~~~~~~~..

16N:,V 9 I.

,:, 1 ' .. ...:.,. -ti .1, :. i-t

1 2 3 MSE 1 2 3 MSE (Iu - 1 rzz eve/lacZ U*r eve/lacZ I 1I1

FIG. 4. The AT2-corepressor site is critical for ventral repression. Embryos are oriented with anterior to the left and dorsal up. Early nuclear cycle 14 embryos contain with two copies of the VRE 200 5' of the MSE eve lacZ reporter. (A) Wild-type zen VRE 200 placed 5' of the MSE eve lacZ fusion gene. The early stripe 2 expression pattern consists of a broad anterior band that is ventrally repressed by the VRE 200 (16). The VRE 200 lacks the general activator sites contained within the VRE 600, so no general dorsal staining is seen. (B) AT2 sites in both copies of the VRE200 have been changed to AT1 sites. Ventral repression of the broad stipe 2 is abolished and staining is equally strong in ventral and dorsal regions. (C) AT1 sites in both copies of the VRE200 have been changed to AT2 sites. Ventral repression of stripe 2 is not affected. (D) Both ATI and AT3 sites in both copies of the VRE200 are replaced with AT2 sites. Ventral repression of stripe 2 is largely retained. Base substitutions were made with mutagenic oligonucleotides as described previously (12). Sequences of mutagenic oligonucleotides are as follows: ATI to AT2, 5'-GTTGCCCCTATGAACGAATATTGATATAAGT-3'; AT2 to ATI, 5'-GGGGCCTATATTTTCTTTGATTGGGTTTC-3'; AT3 to AT2, 5'-AGTTATAGAGTTATGAACGAATATTGATTGGGCGCGTT-3'.

The zen VRE Functions as a Long-Range Specificity of dl-Corepressor Interactions

To determine the range of VRE-mediated repression, we The preceding results indicate that dl-corepressor complexes examined the expression patterns generated by several fusion can block stripe 2 expression when located far from both the promoters (Fig. 3). As shown previously, the 600-bp VRE transcription start site and MSE activators. Thus, this repres- transiently represses the ventral and lateral expression of a sion appears to be distinct from that mediated by the snail, minimal, 480-bp stripe 2 enhancer (Fig. 3A). The embryo Kruppel, and kni repressors. The activities of the zen VRE and shown in Fig. 3A is at the midpoint of nuclear cleavage cycle snail repressor provide an interesting contrast. Both can block 14. At this time, stripe 2 expression is not fully resolved, so that the ventral expression of eve stripe 2; however, in the case of both the anterior and the posterior borders are a bit fuzzy. This snail, the repressor sites must be located quite close to the pattern fully refines during the next 30 min, before the bicoid activator sites within the stripe 2 enhancer (11). In completion of cellularization. However, during this time VRE contrast, as shown above, dl-corepressor complexes can block silencer activity is lost. Consequently, the VRE can be assessed expression even when they map several kilobases from the only at early stages; in fact, a weak dot of staining can be seen stripe 2 activators. Additional experiments lend further sup- in the ventral-most regions of the embryo in Fig. 3A, indicating port to the notion that the VRE is distinct from snail and other that this embryo is just entering the point in development when short-range repressors. the VRE is losing activity. The staining observed along the Kruppel, snail, and kni do not appear to require DNA- dorsal surface is due to general activator binding sites located binding "cofactors" to mediate transcriptional repression. For just distal to the minimal 200-bp VRE contained within the example, a single, 10-bp synthetic Kruppel-binding site is 600-bp fragment used in these assays (17). sufficient to repress the expression of the heterologous rho To determine whether the VRE can function over long NEE in central regions of early embryos (12). It is unlikely that distances, we analyzed the expression of fusion genes contain- this synthetic site contains recognition sequences for both ing the MSE located upstream of the lacZ and Kruppel and additional, unknown cofactors. Similar argu- the VRE placed downstream of lacZ (Fig. 3 B and C). In these ments pertain to snail and kni (10). However, it is possible that experiments, the closest dl-corepressor sites in the VRE map Kruppel and other short-range repressors recruit non-DNA- nearly 5 kb from the MSE activators. Nonetheless, stripe 2 binding . In contrast, dl can function as a repressor expression continues to be repressed in ventral regions of early only when it interacts with "" that bind to neigh- embryos. The repression is nearly comparable to that observed boring sites. when the VRE is placed immediately upstream of the MSE The VRE contains binding sites for a number of proteins, (Fig. 3B, compare withA). Repression is observed whether the suggesting that the repressor may function as a complex. Some VRE is placed in the same 5'-3' orientation as the lacZ of these components may be redundant. One putative core- transcription unit (Fig. 3B) or is placed in an inverted orien- pressor, DSP1 (dorsal switch protein 1), was identified in yeast tation (Fig. 3C). two-hybrid assays (19). DSP-1 binds to a sequence within the Downloaded by guest on September 29, 2021 9312 Colloquium Paper: Cai et al. Proc. Natl. Acad. Sci. USA 93 (1996) VRE and functions together with dl or other rel proteins to repress transcription in assays. DSP1 may be A required for optimal silencing activity, but does not appear to be necessary for VRE activity because minimal VRE se- quences lacking DSP1-binding sites can still mediate ventral repression in transgenic embryos (16). Another protein, NTF- 1 /Elf-1, was recently reported to bind to the ventral repression ,S elements of the zen and dpp promoters. However, as with .,:J.e DSP1, it is still unclear whether the NTF-1/Elf-1 protein itself is involved in VRE activity (20). In addition to these factors, binding assays with crude nuclear extracts from early embryos identified three 3 MSE potential i I .1 1 corepressor sites, AT1, AT2, and AT3, within the 200-bp VRE x2 L::. .. : ( U La) evef laci (16, 17, 21). Gel retardation assays suggest that the AT1 and I 11 AT2 sites may be recognized by distinct proteins. The AT3 site B may not be essential for repression activity (17). We mu- tagenized the VRE to determine the functional relevance of the putative corepressor sites for ventral repression in trans- genic embryos. *R As discussed above, the 600-bp VRE fragment contains both dl-corepressor-binding sites as well as binding sites for ubiq- uitous activators. To analyze repression activity of the VRE in the absence of these activators, we tested transgenes contain- ing two tandem copies of the minimal 200-bp VRE. This element mediates efficient ventral repression of the stripe 2 1 MSE r. enhancer (Fig. 4A). The only staining seen in the embryo is due x 2 .; ;;i -- e ve/ ac Z to the stripe 2 enhancer; there is no general staining observed i along the dorsal surface. To test the importance of individual putative corepressor sites, substitutions were cre- r ated in the AT2 corepressor site (designated "II" in the e

diagrams below the embryos in Fig. 4) to convert it into an AT1 i site. This abolishes the ventral F .X silencing activity of an q otherwise normal VRE (Fig. 4B), so that stripe 2 is expressed J ;> ; l l...J :i. l at equal levels in dorsal and ventral regions (compare with Fig. # 2 b b N; 4A). It is clear that the AT2 site is essential for silencer activity. Additional experiments examined the importance of the t, ;, AT1 site corepressor. Nucleotide substitutions that convert z f f AT1 into AT2 have little or no effect on VRE silencer activity (Fig. 4C, compare withA). As in the previous experiment, two 1 2 MSE F. tandem copies of the modified VRE were placed upstream of I .. ::.1'.; ':.. 1:' the MSE. Stripe 2 is efficiently repressed in ventral regions of I1 III1 eveX ac Z early embryos (Fig. 4C). This result suggests that AT1 site does not convey a unique activity that is separate from AT2; in fact, FIG. 5. Phasing requirements for dl-corepressor interactions. Early it may be dispensable. Conversion of both the AT1 and AT3 nuclear cycle 14 embryos contain two copies of the VRE 200 5' of the sites into the AT2 sequence also has little effect on the VRE MSE eve lacZ reporter gene. (A) Wild-type;i i r zen;.;sVREi]200';placed 5' of the MSE eve-4acZ fusion gene. Early stripe 2 expression shows a broad silencer (Fig. 4D). In this experiment, the three dl-binding sites anterior band that is ventrally repressed by the VRE 200. (This is the are linked to three copies of AT2, rather than to distinct AT1, same embryo as that shown in Fig. 4A). (B) Insertion of five base pairs AT2, and AT3 sequences. Despite this rather dramatic alter- between AT2 and the dl D2 site in both copies of VRE 200 abolishes ation in the organization ofputative corepressors, the modified the ventral repression. Stripe 2 staining is equally intense in the ventral VRE mediates ventral repression of the linked stripe 2 en- and dorsal regions. (C) Insertion of 10 base pairs between AT2 and D2 hancer (Fig. 4D). The extent of repression is nearly comparable partly restores ventral repression. Base insertions were made with to that obtained with the native VRE (compare Fig. 4D with mutagenic oligonucleotides as previously described (12). Sequences of A). These experiments suggest that interactions between dl and mutagenic oligonucleotides are as follows: 5 bp insertion, 5'- ACGAATATTGATTAAGTTGGGTTTCTC-3'; 10 bp insertion, 5'- the AT2 corepressor are particularly critical for VRE function. GAATATTGATTTATGGATGTTGGGTTTCTC-3'. This may be a result of the distinct of the AT2-binding protein. Strict spacing requirements between two factor binding sites can provide evidence that proteins directly interact on the Helical Phasing of dl-Corepressor-Binding Sites DNA. A well-characterized example involves interactions be- Previous studies suggest that there is no exact spacing require- tween the MCM1 and alpha2 regulatory proteins in yeast (22). ment for short-range repressors. Thus, snail, Kruppel, and kni Cooperative DNA-binding interactions depend on a fixed can quench upstream activators located at various distances. spacing of the two factor binding sites. Even subtle changes in The only obvious requirement is that these repressors must spacing abolish interactions between the two proteins. map relatively close to the activators, within 50 to 150 bp. For To determine whether interactions between dl and core- example, a single snail repressor site can repress a modified rho pressors require a fixed spacing, we altered the positioning of NEE in the ventral mesoderm whether located 45 bp or 50 bp the dl 2- and the critical AT2-binding sites. The centers ofAT2- upstream of the closest activator site (ref. 11, and S. Gray, and linked dl 2-binding sites appear to be separated by 10 bp, unpublished observations). These and other results suggest thereby raising the possibility that the two proteins interact on that short-range repressors can block upstream activators the same side of the DNA helix. The insertion of a 5-bp whether located on the same or opposite side of the DNA helix. "spacer" sequence between the AT2- and dl 2-binding sites Downloaded by guest on September 29, 2021 Colloquium Paper: Cai et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9313 abolishes the silencer activity of an otherwise normal 200 bp repression of stripes 2 and 3 is observed when either the VRE (Fig. SB, compare with A). Substantial silencer function stripe 2 enhancer (Fig. 6A) or stripe 3 enhancer (Fig. 6C) is is restored when d12-and AT2-binding sites are separated by a placed upstream of the VR600 sequence. As in the case of 10-bp spacer sequence, restoring the helical phasing (Fig. 5C). fusion promoters containing the VR600 and stripe 2 (see Fig. The demonstration of a strict phasing requirement distin- 3), the ventral repression is transient and observed only in guishes dl-mediated repression from short-range repression. embryos at the early-to-mid stages of nuclear cleavage cycle This phenomenon is reminiscent of the phasing requirement 14. These results indicate that the zen VRE can function as for NF-KB-, ATF-2-, and HMGI(Y)-binding sites in the inter- a dominant silencer and repress multiple enhancers. Sus- feron promoter for normal virus induction (23) and the tained repression in slightly older embryos depends on the absolute dependence on the relative orientation of YY1- relative positioning of the stripe enhancers and VR600. For binding sites for the repression of the c-fos promoter (24). example, stripe 2 repression is maintained when the stripe 2 enhancer is placed upstream of the VRE (Fig. 6B). In The zen VRE Can Silence Multiple Enhancers in contrast, ventral repression begins to be lost by this stage a Modular Promoter when the stripe 2 enhancer is downstream of the VRE (e.g., see Fig. 3A). Similarly, the stripe 3 enhancer is more It is conceivable that dl-corepressor complexes bound to the efficiently repressed when placed upstream of the VRE (Fig. VRE can function over long distances to block the assembly, 6D, compare with C). Thus, it would appear that the VRE recruitment, or function of the basal transcription complex. works best when located between an enhancer and target A prediction of this form of repression, as compared with the promoter. This position dependence is reminiscent of prop- local quenching of upstream activators, is that the VRE can erties of elements, which block or attenuate inter- block the ventral expression of multiple enhancers in a actions of distal, but not proximal, enhancers with target modular promoter. The results presented in Fig. 6 are promoters (25-27). consistent with this type of dominant represson. Fusion promoters were prepared that contain the 600-bp VRE Summary and Conclusions (VR600) placed between the eve stripe 2 and stripe 3 enhancers. Both stripes are repressed in ventral regions of Recent studies in the Drosophila embryo suggest that there early embryos regardless of orientation. Equally efficient may be two basic forms of transcriptional repression: (i) A B

4 VI- ..A

St.2 VR600 St.3 X 7' ' I E v vN v'7 v7 <23> 4> v v \ \x\x\x\z\x\)v 4 ,,,, r X r X r o o r s 1 1 eve/lacZ

C D

St.3 VR600 St.2

eve/lacZ

FIG. 6. Silencing of multiple enhancers by the VRE 600. (A and C) Embryos are in early nuclear cycle 14; (B and D) embryos are in midnuclear cycle 14. Embryos contain transgenes carrying composite elements with a 500 bp eve stripe 3 enhancer (St.3) and the 480 eve stripe 2 (St.2, same as MSE) separated by a VRE 600 element. The three element ensemble was placed 5' of the eve lacZ reporter. (A and B) eve stripe 3 is placed 5' and eve stripe 2 is 3' of the VRE 600. (C and D) The order is reversed, with eve stripe 2 placed 5' and eve stripe 3 placed 3' of the VRE 600. Both stripes are ventrally repressed by the VRE 600 at early stages. In midnuclear cycle 14 embryos, partial is observed as the activity of the VRE 600 wanes. Downloaded by guest on September 29, 2021 9314 Colloquium Paper: Cai et al. Proc. Natl. Acad. Sci. USA 93 (1996)

long-range and (ii) short-range (summarized in Fig. 7). Short- such as eve, a novel repressor site is likely to affect only one range repression permits enhancer autonomy within a com- enhancer, and alter the gene's expression pattern in a subtle plex, modular promoter, whereas long-range repression is manner. In contrast, long-range repression elements, such as dominant, blocking all enhancers. Short-range repressors must those found in the zen and dpp promoters, consist of complex map within 50-150 bp of the upstream activators that they assemblies of several different protein binding sites, which are inhibit (or quench), while the long-range dl2-AT2 corepressor less likely to arise ab initio from a few nucleotide changes in a complex can work over distances of nearly 5 kb. The mecha- promoter region. However, when a novel long-range repressor nism by which long-range repressors function is unknown. The element is introduced near a gene, the expression pattern of dominant, enhancer-nonspecific activity of the zen VRE sug- the gene will be grossly affected. gests that this element may interact directly with the basal machinery to shut down transcription. Long-range repressors We thank Liezelle dela Pena for technical assistance. This work was may block a kinase activity within the basal transcription supported by National Institutes of Health Grant GM 46638. D.N.A. was funded by a postdoctoral fellowship from the American complex, as has been suggested for the yeast TUP1 repressor Society and a National Institutes of Health training grant. (28, 29), or it may stably interact with the basal promoter element, occluding this region so that other enhancers cannot 1. Levine, M. & Manley, J. L. (1989) Cell 59, 405-408. contact the basal promoter region. As additional components 2. Johnson, A. D. (1995) Cell 81, 655-658. of the VRE repression complex are identified, various models 3. Nusslein-Volhard, C., Kluding, H. & Jurgens, G. (1985) Cold can be tested. Spring Harbor Symp. Quant. Biol. 50, 145-154. An important difference between long-range and short- 4. Macdonald, P., Ingham, P. & Struhl, G. (1986) Cell 47, 721-734. range repression may be that long-range repressors induce 5. Frasch, M. & Levine, M. (1987) Genes Dev. 1, 981-995. in stable 6. Small, S., Blair, A. & Levine, M. (1992) EMBO J. 11, 4047-4057. relatively long-lived alterations (or form relatively 7. Small, S., Blair, A. & Levine, M. (1996) Dev. Biol. 175, 314-324. complexes with) the transcription machinery. In contrast, 8. Rothe, M., Nauber, U. & Jackle, H. (1989) EMBO J. 8, 3087- short-range repressors cannot induce such permanent changes, 3094. because this effect would interfere with the activity of neigh- 9. Yan, R. Q., Small, S., Desplan, C., Dearolf, C. R. & Darnell, J. boring enhancers thereby forbidding enhancer autonomy in (1996) Cell 84, 421-430. modular promotor regions. Perhaps the key difference be- 10. Arnosti, D. N., Gray, S., Barolo, S., Zhou, J. & Levine, M. (1996) tween short- and long-range repressors is the stability of their EMBO J. 15, 3659-3666. interactions with the basal transcriptional machinery. 11. Gray, S., Szymanski, P. & Levine, M. (1994) Genes Dev. 8, 1829-1838. The functional differences between short- and long-range 12. Gray, S. & Levine, M. (1996) Genes Dev. 10, 700-710. repressors would allow for distinct forms of evolutionary 13. Small, S., Arnosti, D. N. & Levine, M. (1993) Development change in gene regulation. Because short-range repression can (Cambridge, UK) 119, 767-772. be mediated by a single binding site for a protein, such as snail 14. Studer, M., Popperl, H., Marshall, H., Kuroiwa, A. & Krumlauf, or kni, relatively minor nucleotide changes should therefore be R. (1994) Science 265, 1728-1732. sufficient to introduce a binding site and bring a gene under 15. Ip. Y. T., Kraut, R. Levine, M. & Rushlow, C. A. (1991) Cell 64, control of such a repressor. In a modular promoter for a gene 439-446. 16. Jiang, J., Rushlow, C. A., Zhou, Q., Small, S. & Levine, M. (1992) EMBO J. 11, 3147-3154. Transcriptional Repressor 17. Jiang, J., Cai, H., Zhou, Q. & Levine, M. (1993) EMBO J. 12, short-range long-range 3201-3209. 18. Doyle, H. J., Kraut, R. & Levine, M. (1989) Genes Dev. 3, range of action 50-150 bp > 4 kbp 1515-1533. 19. Lehming, N., Thanos, D., Brickman, J. M., Ma, J., Maniatis, T. & Ptashne, M. (1994) Nature (London) 371, 175-179. DNA-binding single protein multi-protein complex 20. Huang, J.-D., Dubnicoff, T., Liaw, G. J., Bai, Y., Valentine, S. A., components Shirokawa, J. M., Lengyel, J. A. & Courey, A. J. (1996) Genes Dev. 9, 3177-3189. dominant repression no yes 21. Kirov, N., Zhelnin, L., Shah, J. & Rushlow, C. (1993) EMBO J. of multiple enhancers (unless located 12, 3193-3199. near promoter) 22. Vershon, A. K. & Johnson, A. D. (1993) Cell 72, 105-112. 23. Thanos, D. & Maniatis, T. (1992) Cell 71, 777-789. enhancer autonomy yes no 24. Natesan, S. & Gilman, M. Z. (1993) Genes Dev. 7, 2497-2509. 25. Cai, H. & Levine, M. (1995) Nature (London) 376, 533-536. 26. Geyer, P. K. & Corces, V. G. (1992) Genes Dev. 6, 1865-1873. permits yes, when located no 27. Kellum, R. & Schedl, P. (1991) Cell 64, 941-950. promoter autonomy near basal promoter 28. Herschbach, B. M., Arnaud, M. B. & Johnson, A. D. (1994) Nature (London) 370, 309-311. 29. Liao, S. M., Zhang, J., Jeffery, D. A., Koleske, A. J., Thompson, FIG. 7. Functional and regulatory differences between short- and C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J. & Young, long-range repressors. R. A. (1995) Nature (London) 374, 193-196. Downloaded by guest on September 29, 2021