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Control of a Genetic Regulatory Network by a Selector Gene

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Science  11 May 2001:
Vol. 292, Issue 5519, pp. 1164-1167
DOI: 10.1126/science.1058312

Abstract

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It is shown that SCALLOPED, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for SCALLOPED and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development.

The concept of the morphogenetic field, a discrete set of cells in the embryo that gives rise to a particular structure, has held great importance in experimental embryology (1). The discovery of genes whose products control the formation and identity of various fields, dubbed “selector genes” (2), has enabled the recognition and redefinition of fields as discrete territories of selector gene activity (3). Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include theHox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such aseyeless (4), Distal-less(5), and vestigial-scalloped(vg-sd) (6–8), whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks.

In the Drosophila wing imaginal disc, the VG-SD selector protein complex regulates wing formation and identity (7,8). SD is a TEA-domain protein (9) that binds to DNA in a sequence-specific manner (7), whereas VG, a novel nuclear protein (10), functions as a trans-activator (11). To determine whether direct regulation by SD is widely required for gene expression in the wing field, we analyzed the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements (Fig. 1A). We focused in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut(12), spalt (sal) (13), and vg (6).

Figure 1

SD function is required for wing-specific target gene expression. (A) A simplified portion of a genetic regulatory network in the wing disc, including those signaling pathways and target genes analyzed here (highlighted in yellow). (B to I) Gene expression in the wing pouch is dependent upon SD activity. Protein or reporter gene expression in late third larval instar wing imaginal discs is shown (red in B to E, magenta in F to I). Mitotic clones homozygous for the hypomorphicsd58 allele are marked by the absence of the Myc epitope tag (green in F to I) and are circled, and cells are marked by the nuclear dye TOPRO (blue in F to I). (B and F) CUT protein expression along the D-V boundary. (C and G) WG protein expression along the D-V boundary. (D and H) β-Galactosidase expression driven in a broad domain straddling the A-P boundary by the sal10.2-kb element (13), and (E and I) by the vgQ enhancer throughout the wing blade except along the D-V boundary (6). The expression of each candidate target gene is eliminated or reduced by the reduction of SD function.

We first tested whether sd gene function was required for the expression of various genes in the wing field. We generated mitotic clones of cells homozygous for a strong hypomorphic allele ofsd and assessed the expression of gene products or reporter genes within these clones (14). Reduction ofsd function reduced or eliminated the expression of the CUT (Fig. 1, B and F) and WINGLESS (WG) (Fig. 1, C and G) proteins and of reporter genes under the control of the sal 10.2-kb (Fig. 1, D and H) and the vg quadrant (Fig. 1, E and I) enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field.

These results, however, do not distinguish between the direct and indirect regulation of target gene expression by VG-SD. To differentiate between these possibilities, we tested whether the DNA binding domain of SD could bind to specific sequences incut, sal, and vg wing-specific cis-regulatory elements (15). Using DNase I footprinting, we identified SD-binding sites in all of the elements assayed (Fig. 2A) (16). Thus, SD may control the expression of these genes by binding to their cis-regulatory elements.

Figure 2

SD protein binds to and directly regulates the expression of multiple wing-specific cis-regulatory elements in vivo. (A) The topology of sites that bind SD (S; tandem sites denoted S/S), SU(H) (Su), and MAD (M) in the cut,sal, and vg cis-regulatory elements. (B) The sequences of SD-binding sites that have been shown to be required for the activity of wing-specific cis-regulatory elements. Tandem sites have arrows over them. (C) Gel mobility–shift experiments with wild-type and mutated double-stranded oligonucleotide probes spanning tandem SD-binding sites in the cut 0.7-kb and sal 328–base pair (bp) elements. Increasing amounts of SD TEA domain utilized for each probe were, from left to right, 0, 0.1, 0.3, 1, 3, and 10 ng/25 μl. Labels: F, free probe; 1, complex with one SD TEA domain bound; 2, complex with two molecules bound. (D to I) β-Galactosidase expression driven by the (D) cut 0.7-kb, (E) sal328-bp, and (F) vgQ enhancers, respectively, in late third larval instar wing imaginal discs. (G) Expression driven by thecut 0.7-kb enhancer in which the tandem SD-binding sites have been altered. (H) The tandem SD sites in the sal 328-bp element shown in (H) are required for wing expression. (I) Expression driven by the vgQ enhancer, in which four single SD-binding sites have been mutated, is significantly reduced or eliminated.

To determine whether SD binding to these sites was necessary for the function of these cis-regulatory elements in vivo, we mutated specific SD-binding sites within each of the elements such that they reduced or abolished SD binding in gel mobility–shift assays (Fig. 2, B and C) (17). The mutation of tandem SD-binding sites in the cut and sal elements resulted in complete loss of reporter gene expression in vivo (Fig. 2, G and H). Similarly, mutation of the four single SD-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression (Fig. 2I). These results show that SD binds to and directly regulates the expression of four genes—cut, sal, vg, and DSRF (7)—in the wing genetic regulatory network. This molecular analysis and the genetic requirement for SD function for the expression of other genes (18) suggest a widespread requirement for direct VG-SD regulation of genes expressed in the wing field.

Each of the SD targets we analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary (19–21), and thesal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis (22–26). Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [SU(H)], and Mothers Against Dpp (MAD), and Medea (MED), respectively (24, 27,28), have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements (6,24, 29) and occur in these elements (Fig. 2A). This observation, coupled with our data demonstrating a direct requirement for SD binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field.

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, we built a number of synthetic regulatory elements comprised of combinations of binding sites for SD with those for SU(H) or MAD/MED (30). We compared the activity of these elements with those composed of tandem arrays of just selector- or signal effector–binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in nativeDrosophila cis-regulatory elements that have been demonstrated to function in vivo (6, 7,24).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing (Fig. 3, A to C). In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [SD]2 [SU(H)]2 element drove wing-specific expression along the dorsoventral margin (Fig. 3D), consistent with Notch activation along this boundary (19,20, 31), and the [SD]2 [MAD/MED] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc (Fig. 3E), consistent with Dpp-signaling activity along this boundary (32,33). These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of SU(H) and MAD/MED sites were not active in vivo (Fig. 3F), demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the VG-SD complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways.

Figure 3

Combinatorial regulation of gene expression in the wing field. Reporter gene expression driven by synthetic regulatory elements in Drosophila late third larval instar wing imaginal discs. Synthetic elements were composed of the following: (A) two MAD- and two MEDEA-binding sites, (B) four SD-binding sites, (C) four SU(H)-binding sites, (D) two SU(H)-binding sites and two SD-binding sites, (E) a MAD- and a MEDEA-binding site and two SD-binding sites, (F) a MAD- and MEDEA-binding site and two SU(H)-binding sites. MAD- and MEDEA-binding sites are represented by red circles, SU(H)-binding sites by blue triangles, and SD-binding sites by green squares.

There are several potential mechanisms whereby selector proteins and signaling effectors might operate in a combinatorial manner to regulate transcription. One mechanism is through cooperative interactions that increase the occupancy of transcription factor–binding sites on the DNA. Such a scenario appears unlikely in this case, because it would require that each selector protein be able to interact directly with many different signaling pathway transcriptional effectors. Furthermore, cooperative filling of binding sites alone is insufficient to explain selector-signal synergy, because SD alone binds cooperatively to DNA [Fig. 2C and (7)], and yet the presence of multiple SD-binding sites alone is insufficient to generate transcriptional activation.

A second, more likely mechanism underlying selector-signal synergy is the formation of complexes between the two classes of transcription factors and required transcriptional coactivators. Coactivators facilitate transcription by relieving repression by chromatin and/or by mediating interactions with the basal transcriptional machinery (34). We suggest that gene activation by selector proteins and signaling pathways may require both of these activities, and these proteins may form complexes with coactivators on the cis-regulatory DNA (Fig. 4). These complexes could include coactivators such as the multifunctional protein CBP, which has been shown to interact directly with three signaling pathway transcriptional effectors, MAD, CI, and dTCF (35), and also appears to interact with SD (36). Alternatively, synergy between SD and signaling pathway transcriptional effectors could be mediated by different coactivators, with independent functions (Fig. 4B). The obligate requirement for combined inputs from selector genes and signaling pathways, seen here in the wing, may be a general mechanism whereby a universally deployed set of signals can elicit field, tissue, and cell type–specific genetic responses (37).

Figure 4

Models for the field-specific control of transcription by selector proteins and signal effectors. Our results suggest that only when binding sites for both classes of transcription factors are occupied is a functional activation complex assembled and the basal transcriptional apparatus (BTA) enabled. (A) The two transcription factors could bind to different faces of the same coactivator (in blue, e.g., CBP), which could mediate chromatin remodeling via histone acetyltransferase and could mediate interactions with the BTA. (B) Alternatively, transcriptional effectors of signaling pathways and selector proteins may recruit different coactivators (in blue) that perform these functions independently.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: sbcarrol{at}facstaff.wisc.edu

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