REPORTS 。mw v.3.02,Copyright 199 99 m:th ted in Table 1). Control of a Genetic Regulatory Network by a Selector Gene netic regulatory network cor nent. ions of b ng sites to D an egulys.Th egu The concept of the mor ctic field,a dis. scription of many genes distributed throughout then.the SDsclectorprotG to DNA i novel nuclear DP.The the rent wing pattem element d I-spe ctor pr (13 )and vg (6). he ace ere or the sd d.In Prnciplkesckecto 47060 wasega only a fe s thus exerting much of thei for a stro effect indirectly.or they may regulate the trar allele of sd and as W.Mino Methods Enrymol 276 nd F)and WINGLESS (WG)(Fie.1.C 6997 4.Kim.A.T..Acta. ibuted e he vg quadrant (Fig.1.E and I)enhancers 1164 11MAY2001 VOL 292 SCIENCE www.sci .org dithiothreitol (DTT), 10 mM adenosine diphosphate (ADP), and 10% glycerol. Crystallization was initiated by the addition of microseeds, prepared from sub￾strate crystals, after 14 to 20 hours. Plate-like crys￾tals grew in about 10 days. The crystals were har￾vested in solutions containing 20% (w/v) PEG 4000, 0.3 M ammonium acetate, 25 mM Na-Hepes (pH 7.5), 50 mM DTT, 10 mM ADP and 10% glycerol. For cryoprotection, the crystals were transferred to so￾lutions containing increasing glycerol (15, 20, and 25%) for about 1 min each and flash-cooled in liquid propane. Initial data for a rosuvastatin complex struc￾ture to a resolution of 2.4 Å were collected at beam￾line 5.0.2 of the Advanced Light Source (ALS) syn￾chrotron, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Law￾rence Berkeley National Laboratory. Data for the other inhibitor complexes and higher resolution data for the rosuvastatin complex were collected at beam￾line F1 at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation under award DMR-9311772, us￾ing the Macromolecular Diffraction at CHESS (Mac￾CHESS) facility, which is supported by award RR- 01646 from the National Institutes of Health. Data reduction and processing were carried out with the HKL package (15). Because the low-resolution data for the rosuvastatin complex crystal was incomplete for the data collected at CHESS, the reduced data were merged with the reduced data collected at ALS during scaling. All crystals have the symmetry of space group P21 and contain four HMGR monomers in each asymmetric unit, although two different crys￾tal forms were observed ( Table 1). The protein por￾tion of the structure of human HMGR in complex with HMG, CoA, and NADP1 [Protein Data Bank (PDB) code 1dqa] was used as the starting model for the refinement. Initially, the inhibitor molecules were placed into Fo-Fc electron-density maps. Subsequent￾ly, their positions were modified by consulting sA weighted 2Fo-Fc maps (16) and simulated-annealing omit maps (17). The models were built using the program O (18) and refined with CNS (19). Bulk solvent, overall aniosotropic B-factor scaling, and noncrystallographic symmetry restraints were ap￾plied throughout the refinement process. For each of the six HMGR-statin complexes, the electron-density maps were excellent for all four statin molecules bound to the four crystallographically independent monomers. Additionally, poor electron density was located close to residues Y479 and F629 (20) and was interpreted as ADP. The positions of the ADP mole￾cules resemble the positions of the adenosine moi￾eties of the substrates CoA or NADPH. ADP was bound only to some of the CoA or NADPH binding sites and the number of ADP molecules is different for the six structures. 11. C. M. Lawrence, V. W. Rodwell, C. V. Stauffacher, Science 268, 1758 (1995). 12. L. Tabernero, D. A. Bochar, V. W. Rodwell, C. V. Stauffacher, Proc. Natl. Acad. Sci. U.S.A. 96, 7167 (1999). 13. All calculations on accessible or buried surface areas for the statins or the protein, as well as distance information between specific groups, represent aver￾ages for the four crystallographically independent statin molecules observed in each complex structure. The surface accessible areas for the unbound statins, the bound statins, and the buried surface areas upon statin binding to HMGR, respectively, are as follows: compactin 670 Å2, 100 Å2, 880 Å2; simvastatin 670 Å2, 110 Å2, 880 Å2; fluvastatin 660 Å2, 80 Å2, 870 Å2; cerivastatin 720 Å2, 100 Å2, 880 Å2; atorvastatin 840 Å2, 150 Å2, 1060 Å2; and rosuvastatin 710 Å2, 130 Å2, 880 Å2. 14. M. S. Brown, J. R. Faust, J. L. Goldstein, J. Biol. Chem. 253, 1121 (1978). 15. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 306 (1997). 16. A. Hodel, S.-H. Kim, A. T. Bru¨nger, Acta Crystallogr. A 48, 851 (1992). 17. R. J. Read, Acta Crystallogr. A 1986, 140 (1986). 18. T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr. A 47, 110 (1991). 19. A. T. Bru¨nger et al., Acta Crystallogr. D 54, 905 (1998). 20. Single-letter abbreviations for the amino acid resi￾dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 21. G. A. Holdgate et al., in preparation. 22. R. M. Esnouf, Acta Crystallogr. D 55, 938 (1999). 23. L. Esser, personal communication. 24. Persistence of Vision Ray Tracer v.3.02, Copyright 1997, POV-Team. www.povray.org 25. We thank AstraZeneca for providing simvastatin, flu￾vastatin, cerivastatin, atorvastatin, and rosuvastatin and for stimulating discussions; S. Jeong for convert￾ing compactin to the active sodium salt form; the personnel at ALS beamline 5-1 and CHESS beamline F1 for their assistance in data collection; and C. A. Brautigam for critical reading of the manuscript. The coordinates are available from the PDB (accession numbers are indicated in Table 1). 26 January 2001; accepted 3 April 2001 Control of a Genetic Regulatory Network by a Selector Gene Kirsten A. Guss,* Craig E. Nelson,* Angela Hudson, Mary Ellen Kraus, Sean B. Carroll† 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 dis￾crete 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 the Hox genes, whose products differentiate the identity of homolo￾gous fields, and field-specific selector genes such as eyeless (4), Distal-less (5), and vesti￾gial-scalloped (vg-sd) (6–8), whose products have the unique property of directing the for￾mation of entire complex structures. The mech￾anisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector pro￾teins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the tran￾scription 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 ana￾lyzed the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the de￾velopment 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). 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 hypo￾morphic allele ofsd and assessed the expression of gene products or reporter genes within these clones (14). Reduction of sd 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, Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA. *These authors contributed equally to this work. †To whom correspondence should be addressed. E￾mail: sbcarrol@facstaff.wisc.edu R EPORTS 1164 11 MAY 2001 VOL 292 SCIENCE www.sciencemag.org