Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press The Fab-7 element of the bithorax complex attenuates enhancer-promoter mteracnons in the Drosophila embryo Jumin Zhou, 1 Scott Barolo, 2 Paul Szymanski, 3 and Michael Levine 1'4 1Department of Molecular and Cellular Biology, Division of Genetics, University of California, Berkeley, California 94720-3201 USA; ~Departrnent of Biology, University of California at San Diego, La Jolla, California 92093 USA; 3Amplicon, Inc., East Setauket, New York 11733 USA Enhancers integrate positive and negative regulatory information to direct localized patterns of gene expression in the Drosophila embryo. Here we present evidence for the occurrence of cis regulatory elements that control the levels of gene expression by modulating enhancer-promoter interactions. For this purpose we have investigated the Drosophila bithorax complex (BX-C) because genetic studies suggest that the BX-C contains as much as 300 kb of cis regulatory information. A specialized DNA element, Fab-7, has been proposed to function as a boundary element that separates the iab-6 and iab-7 cis regulatory regions within the Abd-B domain of the BX-C. A 1.2-kb Fab-7 DNA fragment was placed between divergently transcribed white and lacZ test promoters and challenged with several defined enhancers expressed in the early embryo. These studies suggest that Fab-7 functions as an attenuator, which weakens gene expression by reducing enhancer-promoter interactions. Fab-7 selectively blocks distal enhancers in an orientation-independent fashion, and can function when located far from either the distal enhancer or target promoter. Fab-7 may be related to insulator DNAs, which flank genetic loci and functionally isolate neighboring genes. We propose that specialized DNA elements, such as the Fab-7 attenuator, might play a general role in controlling the levels of gene expression by modulating enhancer-promoter interactions. [Key Words: Drosophila; Fab-7; enhancer; attenuator] Received August 30, 1996; revised version accepted October 29, 1996. The Drosophila bithorax complex (BX-C) contains three within PS 10, PS 12, and PS 14 (Busturia and Bienz 1993). Hox-containing transcription units, which encode ho- Repression in anterior regions, PS 1 through 9, appears to meotic selector proteins that control the patterning of be initiated by the gap proteins hunchback (hb), Kruppel thoracic and abdominal body segments (Lewis 1978; (Krl, and knirps (kni). This repression is thought to be Sanchez-Herrero et al. 1985; Lewis et al. 1995; Martin et maintained in older embryos (and larvae) by proteins en- al. 1995). One of these, Abdominal-B (Abd-B), controls coded by the Polycomb group (Pc-G; Wedeen et al. 1986; the morphogenesis of posterior regions, including para- Busturia and Bienz 1993; Orlando and Paro 1993; Chan segments (PS) 10 through 14 (Karch et al. 1985; Tiong et et al. 1994; Chiang et al. 1995; Zink and Paro 1995). al. 1985; Casanova et al. 1986). The gene is regulated by It has been proposed that boundary elements separate a series of complex cis regulatory regions, iab-5 through neighboring cis regulatory regions within the Abd-B do- iab-9, that restrict expression within specific paraseg- main (Gyurkovics et al. 1990; Galloni et al. 1993; Karch ments (Karch et al. 1985; Celniker et al. 1989, 1990; et al. 1994). These may function in a manner analogous DeLorenzi and Bienz 1990; Boulet et al. 1991; Sanchez- to insulators, which functionally isolate neighboring Herrero 1991; see Fig. 1). For example, the iab-6 region is genes by blocking interactions of cis elements with in- thought to contain a series of enhancers that mediate appropriate target promoters (Kellum and Schedl 1991, expression within different tissues of PS 11 (e.g., 1992; Geyer and Corces 1992; Dorsett 1993). Previous Vazquez et al. 1993). studies have identified two potential boundary elements, Transcriptional repression is essential for the estab- Mcp and Fab-7 (Gyurkovics et al. 1990; Galloni et al. lishment and maintenance of parasegment-specific pat- 1993; Karch et al. 1994). Mcp resides between iab-4 and terns of Abd-B expression. Previous studies have identi- iab-5, whereas Fab-7 maps between iab-6 and lab-7 fied a 1-kb enhancer, IAB5, that directs expression (summarized in Fig. 1). Mutations in Fab-7 result in the misregulation of Abd-B in PS 11, and a corresponding homeotic transformation of PS 11 tissues (sixth abdom- inal segment; A6) into PS 12 (A7; Gyurkovics et al. 1990; 4Correspondingauthor. Galloni et al. 1993). Abd-B is normally expressed in an GENES & DEVELOPMENT 10:3195-3201 91996 by Cold Spring Harbor LaboratoryPress ISSN 0890-9369/96 $5.00 3195 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Zhou et al. silences the anterior expression of a bxd-Ubx fusion pro- moter that is normally expressed throughout most of the embryonic CNS; this repression is lost in Pc mutants (Busturia and Bienz 1993). In the present study we analyzed the activities of a 1.2-kb Fab-7 DNA fragment in the context of synthetic gene complexes containing defined enhancers expressed in the precellular embryo. We show that this Fab-7 frag- ment selectively blocks or attenuates distal, but not proximal, enhancers. A variety of enhancers were exam- ined, including IAB5, the even-skipped (eve) stripe 3 en- hancer (Small et al. 1996; Yan et al. 1996), the hairy stripe 1 enhancer (Howard and Struhl 1990; Riddihough and Ish-Horowicz 1991) and the rhomboid lateral stripe enhancer (Ip et al. 1992). The latter enhancers were se- Figure 1. Summary of the BX-C. The complex contains three lected to determine whether Fab-7 functions as a spa- Hox transcription units, Ultrabithorax (Ubx), abdominal-A tially localized silencer, or parasegment-specific PRE. (abd-A), and Abdominal-B (Abd-B). The cis regulatory regions Our findings suggest that Fab-7 functions as an attenu- associated with Abd-B are shown below. The leftward arrow ator, possibly in a manner analogous to previously char- indicates the position of the Abd-B transcription start site. The 1-kb IAB5 enhancer is located in a distal region of iab-5, 57 kb acterized insulator DNAs. We discuss the role of special- downstream of the Abd-B transcription start site. The Fab-7 ized DNA elements, such as attenuators, in gene regu- element is located between iab-6 and lab-7. A blow-up of the lation. element is shown below. It contains three DNaseI-hypersensi- tive sites (indicated by shading). The blue horizontal bars indi- cate regions that are deleted in different Fab-7 mutations. For Results example, the Fab-72 allele deletes two of the three hypersensi- A 1.2-kb Fab-7 fragment contains all three major DNaseI tive sites (Karch et al. 1994). The 1.2-kb Fab-7 fragment used in hypersensitive sites identified in previous studies (Karch this study (bottom-most bar) contains all three hypersensitive et al. 1994; summarized in Fig. 1). It resides between the sites. IAB5 enhancer and Abd-B transcription unit (Busturia and Bienz 1993). As shown previously, the IAB5 en- hancer directs a broad band of expression in paraseg- anteroposterior gradient, with highest levels in posterior ments (PS) 10 through 14 of transgenic, precellular em- regions (PS 12, PS 13, and PS 14), and progressively lower bryos (Fig. 2D). This pattern is resolved into three stripes levels in PS 11 and PS 10 (Harding and Levine 1988; (PS 10, PS 12, and PS 14) after cellularization (Busturia Kuziora and McGinnis 1988; Celniker et al. 1989; De- and Bienz 1993). Lorenzi and Bienz 1990; Boulet et al. 1991). Fab-7 mu- tants display similar levels of Abd-B expression in PS 11 and PS 12 (Galloni et al. 1993). Fab-7 attenuates the IAB5 enhancer Previous studies led to several proposals regarding the A fusion promoter containing the IAB5 enhancer and a nature of Fab-7. Perhaps removal of Fab-7 results in the heterologous head stripe enhancer from the hairy pro- ectopic activation of iab-7 enhancers in PS 11 where moter region (HI; Howard and Struhl 1990; Riddihough they are normally silenced by Pc-G repressors (Vazquez and Ish-Horowicz 1991) directs an additive pattern of et al. 1993). According to this view, Fab-7 separates iab-6 expression when placed between divergently transcribed and iab-7 into distinct chromatin (or loop) domains. Al- white and lacZ reporter genes (Fig. 2A, C; see maps below ternatively, it has been suggested that Fab-7 functions as the panels). Altered patterns of expression are observed a silencer element, which attenuates Abd-B expression when the neutral spacer sequence that separates the two in anterior regions, including PS 11 (Busturia and Bienz enhancers is replaced by the Fab-7 element (Fig. 2B, D). 1993). Evidence that Fab-7 is a boundary element is pro- The leftward white gene is expressed within the limits of vided by the observation that a transgene inserted be- the head stripe, whereas expression in the presumptive tween Fab-7 and lab-7 is activated by iab-7 enhancers, abdomen is nearly lost (cf. Fig. 2B and A). The opposite but not by distal iab-5 or iab-6 regulatory elements, in- situation is observed for the rightward lacZ reporter cluding the IAB5 enhancer (Galloni et al. 1993; see Fig. gene. In this case, expression is lost in the head, but 1). In contrast, the notion that Fab-7 functions as a si- normal staining is observed in the abdomen (cf. Fig. 2D lencer, or Polycomb response element (PRE), is suggested and C). by the analysis of transgenic fly strains containing a large These results suggest that Fab-7 selectively blocks the (3.7-kb) Fab-7 DNA fragment attached to various heter- interactions of distal enhancers.