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Short-Range Repression Permits Multiple Enhancers to Function Autonomously Within a Complex Promoter

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Short-Range Repression Permits Multiple Enhancers to Function Autonomously Within a Complex Promoter Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Short-range repression permits multiple enhancers to function autonomously within a complex promoter Susan Gray, Paul Szymanski, and Michael Levine Department of Biology, Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0322 USA Transcriptional repressors play a key role in establishing localized patterns of gene expression in the early Drosophila embryo. Several different modes of repression have been implicated in previous studies, including competition and direct interference with the transcription complex. Here, we present evidence for "quenching," whereby activators and repressors co-occupy neighboring sites in a target promoter, but the repressor blocks the ability of the activator to contact the transcription complex. This study centers on a zinc finger repressor, snail (sna), which represses the expression of neuroectodermal regulatory genes in the presumptive mesoderm. We show that sna can mediate efficient repression when bound 50-100 bp from upstream activator sites. Repression does not depend on proximity of sna-binding sites to the transcription initiation site. sna is not a dedicated repressor but, instead, appears to block disparate activators. We discuss the importance of quenching as a means of permitting separate enhancers to function autonomously within a complex promoter. [Key Words: Transcriptional repressors; gene expression; Drosophila embryo; quenching; enhancers; complex promoter] Received May 16, 1994; revised version accepted June 15, 1994. Transcriptional repression is essential for the establish- to "silencing" (Ip et al. 1991; Huang et al. 1993; Jiang et ment of localized patterns of gene expression in the al. 1993; Kirov et al. 1993, 1994). All three genes are Drosophila embryo. Different modes of repression have repressed in ventral regions of early embryos in response been proposed, including competition, direct repression, to the dorsal (dl) gradient. Repression of the zen pattern and quenching (for review, see Levine and Manley 1989; is mediated by a distal promoter element, the ventral see Fig. 1). Competition has been invoked to account for repression (VR) element, that contains high affinity dl- spatially restricted expression of segmentation genes in binding sites and closely linked corepressor sites. The the early embryo. For example, even-skipped (eve) stripe VR is able to repress the ventral expression of a number 2 expression is regulated by a 480-bp enhancer that con- of different heterologous promoters, including hunch- tains six activator and six repressor sites (Small et al. back (hb), Kr, and eve (Doyle et al. 1989; Ip et al. 1991; 1992). Four of the six activator sites directly overlap four Jiang et al. 1993). Ventral repression is observed even of the repressor sites. The core recognition sequence of when the dl-corepressor-binding sites in the VR map the bicoid (bcd) activator shares a 6 out of 10 match with several kilobases away from the closest activators in the the binding sites of the Krfippel (Kr] repressor (Driever heterologous promoters (Hoch et al. 1990; Ip et al. 1991). and N/isslein-Volhard 1989; Stanojevic et al. 1989; Tre- These observations suggest that the zen VR might act isman and Desplan 1989). These observations prompted directly on the transcription complex. Certain repressors the proposal that Kr defines the posterior stripe 2 border appear to interfere with the transcription complex when by blocking the binding of bcd activators to overlapping bound to proximal regions of target promoters. Among sites (Small et al. 1991, 1992; Stanojevic et al. 1991). A these are the Drosophila homeo domain proteins eve and similar mechanism has been suggested for the regulation engrailed [(en) Han and Manley 1993a, b], as well as the of Kr (Hoch et al. 1992). Competition has also been im- unliganded form of the mammalian thyroid hormone re- plicated in cross-regulatory interactions among mem- ceptor (Fondell et al. 1993) bers of the steroid receptor superfamily (e.g., Liu et al. Here, we present evidence for repression through 1993). "quenching," whereby activators and repressors bind ad- Localized patterns of zerknfillt (zen), decapentaplegic jacent sites, but the repressors mask the ability of the (dpp), and tolloid (tld) expression appear to depend on a activators to contact the transcription complex (see leg- long-range mechanism of direct repression, perhaps akin end to Fig. 1). This study centers on the zinc finger re- GENES & DEVELOPMENT8:1829-1838 91994 by Cold Spring HarborLaboratory Press ISSN 0890-9369/94 $5.00 1829 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Gray et al. gous bcd activator. We discuss the importance of quenching as a means for permitting separate enhancers to function autonomously within a complex promoter. Results sna repressor sites need not overlap dI/HLH activator sites The rho NEE contains four high affinity d/-binding sites, five E boxes [helix-loop-helix (HLH) recognition se- quences], and four sna repressor sites (see legend to Fig. 2). Previous studies have shown that mutations in the sna sites result in the ventral derepression of the rho pattern, so that there is equally intense staining in both Figure 1. Modes of transcriptional repression. The diagrams the lateral neuroectoderm and ventral mesoderm (see summarize three different modes of repression. Competition Fig. 2C, D; Ip et al. 1992). To investigate the mechanism involves the occlusion of activator sites by the binding of over- by which sna represses rho we have introduced synthetic lapping repressors. Direct repression involves interference with sna-binding sites at various positions in and near a de- the formation or activity of the transcription complex (TC). Finally, according to a quenching mechanism, activator and re- fective rho NEE lacking all four native sna repressor pressor co-occupy neighboring sites, but the repressor masks sites. The s2 recognition sequence was used in these ex- the ability of the activator to contact the transcription complex. periments because other sna sites, such as s3 and s4, also include an E-box activator site (Ip et al. 1992; Kasai et al. 1992). Modified NEEs were attached to a lacZ reporter pressor snail (sna), which is a key mesoderm determi- gene and then introduced into embryos via P transfor- nant in the Drosophila embryo (Simpson I983; Boulay et mation. The resulting expression patterns were visual- al. 1987). sna- mutants fail to gastrulate and lack me- ized by whole mount in situ hybridization using a lacZ soderm derivatives such as muscles and connective tis- antisense RNA probe labeled with a digoxigenin-tagged sues (Simpson 1983; Alberga et al. 1991; Leptin 1991). UTP analog (Tautz and Pfeifle 1989; Jiang et al. 1991). sna defines the mesoderm/neuroectoderm boundary of The first set of experiments involved the use of a 700- the early embryo by repressing the expression of neuro- bp DNA fragment that maps between - 2.2 and - 1.5 kb ectodermal regulatory genes in the presumptive meso- upstream of the rho transcription start site. This frag- derm (Kosman et al. 1991; Leptin 1991). sna homologs ment encompasses the entire 300-bp NEE as well as have been implicated in the gastrulation of zebrafish -200 bp of flanking sequence on either side of the NEE. (Hammerschmidt and N~isslein-Volhard 1993; Thisse et The flanking sequences lack high affinity d/, basic HLH al. 1993), frogs (Sargent and Bennett 1990), chicks (Nieto (bHLH), and sna-binding sites (Ip et al. 1992). However, et al. 1994), and mice (Smith et al. 1992), so there is every we cannot exclude the possibility that these sequences expectation that its mechanism of action is evolution- include low affinity sna repressor sites, because gastru- arily conserved. lating embryos carrying the defective 700-bp NEE show The rhomboid (rho) promoter is an early target of sna slightly reduced levels of staining in the invaginating activity (Bier et al. 1990; Ip et al. 1992). It is expressed in mesoderm (Fig. 2D). two lateral stripes that coincide with the presumptive Synthetic sna s2 sites were placed -150 bp upstream neuroectoderm of early embryos, rho stripes are regu- and -120 bp downstream of the closest d/activators in lated by the 300-bp neuroectoderm enhancer element the defective NEE (Fig. 2E, F). This modified NEE fusion (NEE), which contains d/- and HLH-binding sites that gene, NEE-150, directs an abnormal pattern of expres- activate expression in both the lateral neuroectoderm sion in precellular embryos (Fig. 2E). Intense expression and ventral mesoderm. Expression is excluded from the is detected in both the lateral neuroectoderm and ventral mesoderm by closely linked sna repressor sites. In sna- mesoderm, although there may be a slight attenuation in mutants, the rho expression pattern is completely dere- the levels of staining in the mesoderm. This pattern is pressed and spans both the lateral neuroectoderm and quite similar to that observed for the unmodified NEE ventral mesoderm (Kosman et al. 1991). (of. Fig. 2C). However, by the onset of gastrulation the We present evidence that sna-binding sites need not NEE-150 fusion gene is slightly, but consistently, re- overlap activator sites to repress rho expression in the pressed in the mesoderm (Fig. 2F). This repression is ventral mesoderm. Repression is observed when sna more pronounced than the variably reduced ventral sites map 50 or 120 bp from the closest dl activator sites, staining observed with the comparable NEE lacking syn- although better repression occurs when the sna sites are thetic sna sites (cf. Fig. 2D). It would appear that two sna 50 bp from the activators. Efficient repression is not cor- sites, bracketing the defective NEE, can mediate slight related with the proximity of the sna sites to the tran- repression when they map quite far (120--150 bp) from scription initiation site.
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