Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1992 Nov;11(11):4059–4072. doi: 10.1002/j.1460-2075.1992.tb05499.x

Differential DNA sequence recognition is a determinant of specificity in homeotic gene action.

S C Ekker 1, D P von Kessler 1, P A Beachy 1
PMCID: PMC556916  PMID: 1356765

Abstract

The homeotic genes of Drosophila encode transcriptional regulatory proteins that specify distinct segment identities. Previous studies have implicated the homeodomain as a major determinant of biological specificity within these proteins, but have not established the physical basis of this specificity. We show here that the homeodomains encoded by the Ultrabithorax and Deformed homeotic genes bind optimally to distinct DNA sequences and have mapped the determinants responsible for differential recognition. We further show that relative transactivation by these two proteins in a simple in vivo system can differ by nearly two orders of magnitude. Such differences in DNA sequence recognition and target activation provide a biochemical basis for at least part of the biological specificity of homeotic gene action.

Full text

PDF
4070

Images in this article

Selected References

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

  1. Beachy P. A., Helfand S. L., Hogness D. S. Segmental distribution of bithorax complex proteins during Drosophila development. Nature. 1985 Feb 14;313(6003):545–551. doi: 10.1038/313545a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bergson C., McGinnis W. An autoregulatory enhancer element of the Drosophila homeotic gene Deformed. EMBO J. 1990 Dec;9(13):4287–4297. doi: 10.1002/j.1460-2075.1990.tb07877.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blackwell T. K., Weintraub H. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science. 1990 Nov 23;250(4984):1104–1110. doi: 10.1126/science.2174572. [DOI] [PubMed] [Google Scholar]
  5. Cribbs D. L., Pultz M. A., Johnson D., Mazzulla M., Kaufman T. C. Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia. EMBO J. 1992 Apr;11(4):1437–1449. doi: 10.1002/j.1460-2075.1992.tb05188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dearolf C. R., Topol J., Parker C. S. The caudal gene product is a direct activator of fushi tarazu transcription during Drosophila embryogenesis. Nature. 1989 Sep 28;341(6240):340–343. doi: 10.1038/341340a0. [DOI] [PubMed] [Google Scholar]
  7. Desplan C., Theis J., O'Farrell P. H. The sequence specificity of homeodomain-DNA interaction. Cell. 1988 Sep 23;54(7):1081–1090. doi: 10.1016/0092-8674(88)90123-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dessain S., Gross C. T., Kuziora M. A., McGinnis W. Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos. EMBO J. 1992 Mar;11(3):991–1002. doi: 10.1002/j.1460-2075.1992.tb05138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Diederich R. J., Merrill V. K., Pultz M. A., Kaufman T. C. Isolation, structure, and expression of labial, a homeotic gene of the Antennapedia Complex involved in Drosophila head development. Genes Dev. 1989 Mar;3(3):399–414. doi: 10.1101/gad.3.3.399. [DOI] [PubMed] [Google Scholar]
  10. Driever W., Nüsslein-Volhard C. The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo. Nature. 1989 Jan 12;337(6203):138–143. doi: 10.1038/337138a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fire A., Harrison S. W., Dixon D. A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene. 1990 Sep 14;93(2):189–198. doi: 10.1016/0378-1119(90)90224-f. [DOI] [PubMed] [Google Scholar]
  13. Fitzpatrick V. D., Ingles C. J. The Drosophila fushi tarazu polypeptide is a DNA-binding transcriptional activator in yeast cells. Nature. 1989 Feb 16;337(6208):666–668. doi: 10.1038/337666a0. [DOI] [PubMed] [Google Scholar]
  14. Florence B., Handrow R., Laughon A. DNA-binding specificity of the fushi tarazu homeodomain. Mol Cell Biol. 1991 Jul;11(7):3613–3623. doi: 10.1128/mcb.11.7.3613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gavis E. R., Hogness D. S. Phosphorylation, expression and function of the Ultrabithorax protein family in Drosophila melanogaster. Development. 1991 Aug;112(4):1077–1093. doi: 10.1242/dev.112.4.1077. [DOI] [PubMed] [Google Scholar]
  16. Gibson G., Schier A., LeMotte P., Gehring W. J. The specificities of Sex combs reduced and Antennapedia are defined by a distinct portion of each protein that includes the homeodomain. Cell. 1990 Sep 21;62(6):1087–1103. doi: 10.1016/0092-8674(90)90386-s. [DOI] [PubMed] [Google Scholar]
  17. Graham A., Papalopulu N., Krumlauf R. The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell. 1989 May 5;57(3):367–378. doi: 10.1016/0092-8674(89)90912-4. [DOI] [PubMed] [Google Scholar]
  18. Guazzi S., Price M., De Felice M., Damante G., Mattei M. G., Di Lauro R. Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J. 1990 Nov;9(11):3631–3639. doi: 10.1002/j.1460-2075.1990.tb07574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hafen E., Levine M., Gehring W. J. Regulation of Antennapedia transcript distribution by the bithorax complex in Drosophila. Nature. 1984 Jan 19;307(5948):287–289. doi: 10.1038/307287a0. [DOI] [PubMed] [Google Scholar]
  20. Hanes S. D., Brent R. A genetic model for interaction of the homeodomain recognition helix with DNA. Science. 1991 Jan 25;251(4992):426–430. doi: 10.1126/science.1671176. [DOI] [PubMed] [Google Scholar]
  21. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hoey T., Levine M. Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature. 1988 Apr 28;332(6167):858–861. doi: 10.1038/332858a0. [DOI] [PubMed] [Google Scholar]
  24. Kenyon C., Wang B. A cluster of Antennapedia-class homeobox genes in a nonsegmented animal. Science. 1991 Aug 2;253(5019):516–517. doi: 10.1126/science.1677487. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Kuziora M. A., McGinnis W. A homeodomain substitution changes the regulatory specificity of the deformed protein in Drosophila embryos. Cell. 1989 Nov 3;59(3):563–571. doi: 10.1016/0092-8674(89)90039-1. [DOI] [PubMed] [Google Scholar]
  28. Kuziora M. A., McGinnis W. Altering the regulatory targets of the Deformed protein in Drosophila embryos by substituting the Abdominal-B homeodomain. Mech Dev. 1990 Dec;33(1):83–93. doi: 10.1016/0925-4773(90)90137-b. [DOI] [PubMed] [Google Scholar]
  29. Laughon A. DNA binding specificity of homeodomains. Biochemistry. 1991 Dec 3;30(48):11357–11367. doi: 10.1021/bi00112a001. [DOI] [PubMed] [Google Scholar]
  30. Lin L., McGinnis W. Mapping functional specificity in the Dfd and Ubx homeo domains. Genes Dev. 1992 Jun;6(6):1071–1081. doi: 10.1101/gad.6.6.1071. [DOI] [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. Malicki J., Schughart K., McGinnis W. Mouse Hox-2.2 specifies thoracic segmental identity in Drosophila embryos and larvae. Cell. 1990 Nov 30;63(5):961–967. doi: 10.1016/0092-8674(90)90499-5. [DOI] [PubMed] [Google Scholar]
  33. Mann R. S., Hogness D. S. Functional dissection of Ultrabithorax proteins in D. melanogaster. Cell. 1990 Feb 23;60(4):597–610. doi: 10.1016/0092-8674(90)90663-y. [DOI] [PubMed] [Google Scholar]
  34. McGinnis N., Kuziora M. A., McGinnis W. Human Hox-4.2 and Drosophila deformed encode similar regulatory specificities in Drosophila embryos and larvae. Cell. 1990 Nov 30;63(5):969–976. doi: 10.1016/0092-8674(90)90500-e. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. O'Connor M. B., Binari R., Perkins L. A., Bender W. Alternative RNA products from the Ultrabithorax domain of the bithorax complex. EMBO J. 1988 Feb;7(2):435–445. doi: 10.1002/j.1460-2075.1988.tb02831.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Otting G., Qian Y. Q., Müller M., Affolter M., Gehring W., Wüthrich K. Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif. EMBO J. 1988 Dec 20;7(13):4305–4309. doi: 10.1002/j.1460-2075.1988.tb03329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  39. Percival-Smith A., Müller M., Affolter M., Gehring W. J. The interaction with DNA of wild-type and mutant fushi tarazu homeodomains. EMBO J. 1990 Dec;9(12):3967–3974. doi: 10.1002/j.1460-2075.1990.tb07617.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  41. Regulski M., Dessain S., McGinnis N., McGinnis W. High-affinity binding sites for the Deformed protein are required for the function of an autoregulatory enhancer of the Deformed gene. Genes Dev. 1991 Feb;5(2):278–286. doi: 10.1101/gad.5.2.278. [DOI] [PubMed] [Google Scholar]
  42. Regulski M., Harding K., Kostriken R., Karch F., Levine M., McGinnis W. Homeo box genes of the Antennapedia and bithorax complexes of Drosophila. Cell. 1985 Nov;43(1):71–80. doi: 10.1016/0092-8674(85)90013-3. [DOI] [PubMed] [Google Scholar]
  43. Regulski M., McGinnis N., Chadwick R., McGinnis W. Developmental and molecular analysis of Deformed; a homeotic gene controlling Drosophila head development. EMBO J. 1987 Mar;6(3):767–777. doi: 10.1002/j.1460-2075.1987.tb04819.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  45. Rosenfeld M. G. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev. 1991 Jun;5(6):897–907. doi: 10.1101/gad.5.6.897. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sauer R. T., Smith D. L., Johnson A. D. Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free. Genes Dev. 1988 Jul;2(7):807–816. doi: 10.1101/gad.2.7.807. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Smith D. L., Johnson A. D. A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer. Cell. 1992 Jan 10;68(1):133–142. doi: 10.1016/0092-8674(92)90212-u. [DOI] [PubMed] [Google Scholar]
  51. Struhl G., Struhl K., Macdonald P. M. The gradient morphogen bicoid is a concentration-dependent transcriptional activator. Cell. 1989 Jun 30;57(7):1259–1273. doi: 10.1016/0092-8674(89)90062-7. [DOI] [PubMed] [Google Scholar]
  52. Treisman J., Gönczy P., Vashishtha M., Harris E., Desplan C. A single amino acid can determine the DNA binding specificity of homeodomain proteins. Cell. 1989 Nov 3;59(3):553–562. doi: 10.1016/0092-8674(89)90038-x. [DOI] [PubMed] [Google Scholar]
  53. Treisman J., Harris E., Desplan C. The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev. 1991 Apr;5(4):594–604. doi: 10.1101/gad.5.4.594. [DOI] [PubMed] [Google Scholar]
  54. West R. W., Jr, Yocum R. R., Ptashne M. Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. Mol Cell Biol. 1984 Nov;4(11):2467–2478. doi: 10.1128/mcb.4.11.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wolberger C., Vershon A. K., Liu B., Johnson A. D., Pabo C. O. Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions. Cell. 1991 Nov 1;67(3):517–528. doi: 10.1016/0092-8674(91)90526-5. [DOI] [PubMed] [Google Scholar]
  56. Wysocka-Diller J. W., Aisemberg G. O., Baumgarten M., Levine M., Macagno E. R. Characterization of a homologue of bithorax-complex genes in the leech Hirudo medicinalis. Nature. 1989 Oct 26;341(6244):760–763. doi: 10.1038/341760a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES