Skip to main content
Genetics logoLink to Genetics
. 1995 Jan;139(1):349–363. doi: 10.1093/genetics/139.1.349

Identification of Target Genes Regulated by Homeotic Proteins in Drosophila Melanogaster through Genetic Selection of Ultrabithorax Protein-Binding Sites in Yeast

G S Mastick 1, R McKay 1, T Oligino 1, K Donovan 1, A J Lopez 1
PMCID: PMC1206331  PMID: 7705635

Abstract

A method based on the transcriptional activation of a selectable reporter in yeast cells was used to identify genes regulated by the Ultrabithorax homeoproteins in Drosophila melanogaster. Fifty-three DNA fragments that can mediate activation by UBX isoform Ia in this test were recovered after screening 15% of the Drosophila genome. Half of these fragments represent single-copy sequences in the genome. Six single-copy fragments were investigated in detail, and each was found to reside near a transcription unit whose expression in the embryo is segmentally modulated as expected for targets of homeotic genes. Four of these putative target genes are expressed in patterns that suggest roles in the development of regional specializations within mesoderm derivatives; in three cases these expression patterns depend on Ultrabithorax function. Extrapolation from this pilot study indicates that 85-170 candidate target genes can be identified by screening the entire Drosophila genome with UBX isoform Ia. With appropriate modifications, this approach should be applicable to other transcriptional regulators in diverse organisms.

Full Text

The Full Text of this article is available as a PDF (10.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abel T., Michelson A. M., Maniatis T. A Drosophila GATA family member that binds to Adh regulatory sequences is expressed in the developing fat body. Development. 1993 Nov;119(3):623–633. doi: 10.1242/dev.119.3.623. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Andrew D. J., Scott M. P. Downstream of the homeotic genes. New Biol. 1992 Jan;4(1):5–15. [PubMed] [Google Scholar]
  4. Artero R. D., Akam M., Pérez-Alonso M. Oligonucleotide probes detect splicing variants in situ in Drosophila embryos. Nucleic Acids Res. 1992 Nov 11;20(21):5687–5690. doi: 10.1093/nar/20.21.5687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baker N. E., Mlodzik M., Rubin G. M. Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science. 1990 Dec 7;250(4986):1370–1377. doi: 10.1126/science.2175046. [DOI] [PubMed] [Google Scholar]
  6. Beachy P. A., Krasnow M. A., Gavis E. R., Hogness D. S. An Ultrabithorax protein binds sequences near its own and the Antennapedia P1 promoters. Cell. 1988 Dec 23;55(6):1069–1081. doi: 10.1016/0092-8674(88)90251-6. [DOI] [PubMed] [Google Scholar]
  7. Beachy P. A., Varkey J., Young K. E., von Kessler D. P., Sun B. I., Ekker S. C. Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites. Mol Cell Biol. 1993 Nov;13(11):6941–6956. doi: 10.1128/mcb.13.11.6941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bender W., Akam M., Karch F., Beachy P. A., Peifer M., Spierer P., Lewis E. B., Hogness D. S. Molecular Genetics of the Bithorax Complex in Drosophila melanogaster. Science. 1983 Jul 1;221(4605):23–29. doi: 10.1126/science.221.4605.23. [DOI] [PubMed] [Google Scholar]
  9. Bienz M., Tremml G. Domain of Ultrabithorax expression in Drosophila visceral mesoderm from autoregulation and exclusion. Nature. 1988 Jun 9;333(6173):576–578. doi: 10.1038/333576a0. [DOI] [PubMed] [Google Scholar]
  10. Botas J. Control of morphogenesis and differentiation by HOM/Hox genes. Curr Opin Cell Biol. 1993 Dec;5(6):1015–1022. doi: 10.1016/0955-0674(93)90086-6. [DOI] [PubMed] [Google Scholar]
  11. Brookman J. J., Toosy A. T., Shashidhara L. S., White R. A. The 412 retrotransposon and the development of gonadal mesoderm in Drosophila. Development. 1992 Dec;116(4):1185–1192. doi: 10.1242/dev.116.4.1185. [DOI] [PubMed] [Google Scholar]
  12. Capovilla M., Brandt M., Botas J. Direct regulation of decapentaplegic by Ultrabithorax and its role in Drosophila midgut morphogenesis. Cell. 1994 Feb 11;76(3):461–475. doi: 10.1016/0092-8674(94)90111-2. [DOI] [PubMed] [Google Scholar]
  13. Cavarec L., Heidmann T. The Drosophila copia retrotransposon contains binding sites for transcriptional regulation by homeoproteins. Nucleic Acids Res. 1993 Nov 11;21(22):5041–5049. doi: 10.1093/nar/21.22.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chan S. K., Jaffe L., Capovilla M., Botas J., Mann R. S. The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell. 1994 Aug 26;78(4):603–615. doi: 10.1016/0092-8674(94)90525-8. [DOI] [PubMed] [Google Scholar]
  15. Cumberledge S., Szabad J., Sakonju S. Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster. Development. 1992 Jun;115(2):395–402. doi: 10.1242/dev.115.2.395. [DOI] [PubMed] [Google Scholar]
  16. Ekker S. C., Young K. E., von Kessler D. P., Beachy P. A. Optimal DNA sequence recognition by the Ultrabithorax homeodomain of Drosophila. EMBO J. 1991 May;10(5):1179–1186. doi: 10.1002/j.1460-2075.1991.tb08058.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ekker S. C., von Kessler D. P., Beachy P. A. Differential DNA sequence recognition is a determinant of specificity in homeotic gene action. EMBO J. 1992 Nov;11(11):4059–4072. doi: 10.1002/j.1460-2075.1992.tb05499.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gould A. P., Brookman J. J., Strutt D. I., White R. A. Targets of homeotic gene control in Drosophila. Nature. 1990 Nov 22;348(6299):308–312. doi: 10.1038/348308a0. [DOI] [PubMed] [Google Scholar]
  19. Gould A. P., White R. A. Connectin, a target of homeotic gene control in Drosophila. Development. 1992 Dec;116(4):1163–1174. doi: 10.1242/dev.116.4.1163. [DOI] [PubMed] [Google Scholar]
  20. Graba Y., Aragnol D., Laurenti P., Garzino V., Charmot D., Berenger H., Pradel J. Homeotic control in Drosophila; the scabrous gene is an in vivo target of Ultrabithorax proteins. EMBO J. 1992 Sep;11(9):3375–3384. doi: 10.1002/j.1460-2075.1992.tb05416.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hayashi S., Scott M. P. What determines the specificity of action of Drosophila homeodomain proteins? Cell. 1990 Nov 30;63(5):883–894. doi: 10.1016/0092-8674(90)90492-w. [DOI] [PubMed] [Google Scholar]
  22. Hayflick J. S., Wolfgang W. J., Forte M. A., Thomas G. A unique Kex2-like endoprotease from Drosophila melanogaster is expressed in the central nervous system during early embryogenesis. J Neurosci. 1992 Mar;12(3):705–717. doi: 10.1523/JNEUROSCI.12-03-00705.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hinz U., Wolk A., Renkawitz-Pohl R. Ultrabithorax is a regulator of beta 3 tubulin expression in the Drosophila visceral mesoderm. Development. 1992 Nov;116(3):543–554. doi: 10.1242/dev.116.3.543. [DOI] [PubMed] [Google Scholar]
  24. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  25. Immerglück K., Lawrence P. A., Bienz M. Induction across germ layers in Drosophila mediated by a genetic cascade. Cell. 1990 Jul 27;62(2):261–268. doi: 10.1016/0092-8674(90)90364-k. [DOI] [PubMed] [Google Scholar]
  26. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kaufman T. C., Seeger M. A., Olsen G. Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster. Adv Genet. 1990;27:309–362. doi: 10.1016/s0065-2660(08)60029-2. [DOI] [PubMed] [Google Scholar]
  28. Kissinger C. R., Liu B. S., Martin-Blanco E., Kornberg T. B., Pabo C. O. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions. Cell. 1990 Nov 2;63(3):579–590. doi: 10.1016/0092-8674(90)90453-l. [DOI] [PubMed] [Google Scholar]
  29. Kornfeld K., Saint R. B., Beachy P. A., Harte P. J., Peattie D. A., Hogness D. S. Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila. Genes Dev. 1989 Feb;3(2):243–258. doi: 10.1101/gad.3.2.243. [DOI] [PubMed] [Google Scholar]
  30. Laird C. D., McCarthy B. J. Molecular characterization of the Drosophila genome. Genetics. 1969 Dec;63(4):865–882. doi: 10.1093/genetics/63.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lopez A. J., Hogness D. S. Immunochemical dissection of the Ultrabithorax homeoprotein family in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9924–9928. doi: 10.1073/pnas.88.22.9924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Macias A., Casanova J., Morata G. Expression and regulation of the abd-A gene of Drosophila. Development. 1990 Dec;110(4):1197–1207. doi: 10.1242/dev.110.4.1197. [DOI] [PubMed] [Google Scholar]
  33. McGinnis W., Krumlauf R. Homeobox genes and axial patterning. Cell. 1992 Jan 24;68(2):283–302. doi: 10.1016/0092-8674(92)90471-n. [DOI] [PubMed] [Google Scholar]
  34. Mlodzik M., Baker N. E., Rubin G. M. Isolation and expression of scabrous, a gene regulating neurogenesis in Drosophila. Genes Dev. 1990 Nov;4(11):1848–1861. doi: 10.1101/gad.4.11.1848. [DOI] [PubMed] [Google Scholar]
  35. Nose A., Mahajan V. B., Goodman C. S. Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila. Cell. 1992 Aug 21;70(4):553–567. doi: 10.1016/0092-8674(92)90426-d. [DOI] [PubMed] [Google Scholar]
  36. O'Connell P. O., Rosbash M. Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res. 1984 Jul 11;12(13):5495–5513. doi: 10.1093/nar/12.13.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Padgett R. W., St Johnston R. D., Gelbart W. M. A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature. 1987 Jan 1;325(6099):81–84. doi: 10.1038/325081a0. [DOI] [PubMed] [Google Scholar]
  38. Panganiban G. E., Reuter R., Scott M. P., Hoffmann F. M. A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis. Development. 1990 Dec;110(4):1041–1050. doi: 10.1242/dev.110.4.1041. [DOI] [PubMed] [Google Scholar]
  39. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peifer M., Wieschaus E. Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity. Genes Dev. 1990 Jul;4(7):1209–1223. doi: 10.1101/gad.4.7.1209. [DOI] [PubMed] [Google Scholar]
  41. Pultz M. A., Diederich R. J., Cribbs D. L., Kaufman T. C. The proboscipedia locus of the Antennapedia complex: a molecular and genetic analysis. Genes Dev. 1988 Jul;2(7):901–920. doi: 10.1101/gad.2.7.901. [DOI] [PubMed] [Google Scholar]
  42. Rauskolb C., Peifer M., Wieschaus E. extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene pbx1. Cell. 1993 Sep 24;74(6):1101–1112. doi: 10.1016/0092-8674(93)90731-5. [DOI] [PubMed] [Google Scholar]
  43. Reuter R., Panganiban G. E., Hoffmann F. M., Scott M. P. Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos. Development. 1990 Dec;110(4):1031–1040. doi: 10.1242/dev.110.4.1031. [DOI] [PubMed] [Google Scholar]
  44. Reuter R., Scott M. P. Expression and function of the homoeotic genes Antennapedia and Sex combs reduced in the embryonic midgut of Drosophila. Development. 1990 Jun;109(2):289–303. doi: 10.1242/dev.109.2.289. [DOI] [PubMed] [Google Scholar]
  45. Rizki T. M., Rizki R. M. Larval adipose tissue of homoeotic bithorax mutants of Drosophila. Dev Biol. 1978 Aug;65(2):476–482. doi: 10.1016/0012-1606(78)90042-8. [DOI] [PubMed] [Google Scholar]
  46. Roebroek A. J., Creemers J. W., Pauli I. G., Bogaert T., Van de Ven W. J. Generation of structural and functional diversity in furin-like proteins in Drosophila melanogaster by alternative splicing of the Dfur1 gene. EMBO J. 1993 May;12(5):1853–1870. doi: 10.1002/j.1460-2075.1993.tb05834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Roebroek A. J., Pauli I. G., Zhang Y., van de Ven W. J. cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin. FEBS Lett. 1991 Sep 9;289(2):133–137. doi: 10.1016/0014-5793(91)81052-a. [DOI] [PubMed] [Google Scholar]
  48. Samson M. L., Jackson-Grusby L., Brent R. Gene activation and DNA binding by Drosophila Ubx and abd-A proteins. Cell. 1989 Jun 16;57(6):1045–1052. doi: 10.1016/0092-8674(89)90342-5. [DOI] [PubMed] [Google Scholar]
  49. Scott M. P., Tamkun J. W., Hartzell G. W., 3rd The structure and function of the homeodomain. Biochim Biophys Acta. 1989 Jul 28;989(1):25–48. doi: 10.1016/0304-419x(89)90033-4. [DOI] [PubMed] [Google Scholar]
  50. Tautz D., Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989 Aug;98(2):81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]
  51. Tremml G., Bienz M. Homeotic gene expression in the visceral mesoderm of Drosophila embryos. EMBO J. 1989 Sep;8(9):2677–2685. doi: 10.1002/j.1460-2075.1989.tb08408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wagner-Bernholz J. T., Wilson C., Gibson G., Schuh R., Gehring W. J. Identification of target genes of the homeotic gene Antennapedia by enhancer detection. Genes Dev. 1991 Dec;5(12B):2467–2480. doi: 10.1101/gad.5.12b.2467. [DOI] [PubMed] [Google Scholar]
  53. White R. A., Wilcox M. Distribution of Ultrabithorax proteins in Drosophila. EMBO J. 1985 Aug;4(8):2035–2043. doi: 10.1002/j.1460-2075.1985.tb03889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wilson C., Bellen H. J., Gehring W. J. Position effects on eukaryotic gene expression. Annu Rev Cell Biol. 1990;6:679–714. doi: 10.1146/annurev.cb.06.110190.003335. [DOI] [PubMed] [Google Scholar]
  55. Wilson T. E., Fahrner T. J., Johnston M., Milbrandt J. Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science. 1991 May 31;252(5010):1296–1300. doi: 10.1126/science.1925541. [DOI] [PubMed] [Google Scholar]
  56. van Dijk M. A., Murre C. extradenticle raises the DNA binding specificity of homeotic selector gene products. Cell. 1994 Aug 26;78(4):617–624. doi: 10.1016/0092-8674(94)90526-6. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES