Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Apr;16(4):1632–1640. doi: 10.1128/mcb.16.4.1632

Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex.

Q Lu 1, M P Kamps 1
PMCID: PMC231149  PMID: 8657138

Abstract

Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.

Full Text

The Full Text of this article is available as a PDF (477.9 KB).

Selected References

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

  1. Akam M. Hox and HOM: homologous gene clusters in insects and vertebrates. Cell. 1989 May 5;57(3):347–349. doi: 10.1016/0092-8674(89)90909-4. [DOI] [PubMed] [Google Scholar]
  2. Ananthan J., Baler R., Morrissey D., Zuo J., Lan Y., Weir M., Voellmy R. Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein. Mol Cell Biol. 1993 Mar;13(3):1599–1609. doi: 10.1128/mcb.13.3.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Appel B., Sakonju S. Cell-type-specific mechanisms of transcriptional repression by the homeotic gene products UBX and ABD-A in Drosophila embryos. EMBO J. 1993 Mar;12(3):1099–1109. doi: 10.1002/j.1460-2075.1993.tb05751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bürglin T. R., Ruvkun G. New motif in PBX genes. Nat Genet. 1992 Aug;1(5):319–320. doi: 10.1038/ng0892-319. [DOI] [PubMed] [Google Scholar]
  5. Catron K. M., Zhang H., Marshall S. C., Inostroza J. A., Wilson J. M., Abate C. Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites. Mol Cell Biol. 1995 Feb;15(2):861–871. doi: 10.1128/mcb.15.2.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chang C. P., Shen W. F., Rozenfeld S., Lawrence H. J., Largman C., Cleary M. L. Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev. 1995 Mar 15;9(6):663–674. doi: 10.1101/gad.9.6.663. [DOI] [PubMed] [Google Scholar]
  8. Dedera D. A., Waller E. K., LeBrun D. P., Sen-Majumdar A., Stevens M. E., Barsh G. S., Cleary M. L. Chimeric homeobox gene E2A-PBX1 induces proliferation, apoptosis, and malignant lymphomas in transgenic mice. Cell. 1993 Sep 10;74(5):833–843. doi: 10.1016/0092-8674(93)90463-z. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Fitzpatrick V. D., Percival-Smith A., Ingles C. J., Krause H. M. Homeodomain-independent activity of the fushi tarazu polypeptide in Drosophila embryos. Nature. 1992 Apr 16;356(6370):610–612. doi: 10.1038/356610a0. [DOI] [PubMed] [Google Scholar]
  12. Fortini M. E., Lai Z. C., Rubin G. M. The Drosophila zfh-1 and zfh-2 genes encode novel proteins containing both zinc-finger and homeodomain motifs. Mech Dev. 1991 Jun;34(2-3):113–122. doi: 10.1016/0925-4773(91)90048-b. [DOI] [PubMed] [Google Scholar]
  13. Gehring W. J., Affolter M., Bürglin T. Homeodomain proteins. Annu Rev Biochem. 1994;63:487–526. doi: 10.1146/annurev.bi.63.070194.002415. [DOI] [PubMed] [Google Scholar]
  14. Gehring W. J., Qian Y. Q., Billeter M., Furukubo-Tokunaga K., Schier A. F., Resendez-Perez D., Affolter M., Otting G., Wüthrich K. Homeodomain-DNA recognition. Cell. 1994 Jul 29;78(2):211–223. doi: 10.1016/0092-8674(94)90292-5. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Goutte C., Johnson A. D. Recognition of a DNA operator by a dimer composed of two different homeodomain proteins. EMBO J. 1994 Mar 15;13(6):1434–1442. doi: 10.1002/j.1460-2075.1994.tb06397.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grueneberg D. A., Natesan S., Alexandre C., Gilman M. Z. Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor. Science. 1992 Aug 21;257(5073):1089–1095. doi: 10.1126/science.257.5073.1089. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Hou J., Schindler U., Henzel W. J., Ho T. C., Brasseur M., McKnight S. L. An interleukin-4-induced transcription factor: IL-4 Stat. Science. 1994 Sep 16;265(5179):1701–1706. doi: 10.1126/science.8085155. [DOI] [PubMed] [Google Scholar]
  21. Ingraham H. A., Flynn S. E., Voss J. W., Albert V. R., Kapiloff M. S., Wilson L., Rosenfeld M. G. The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions. Cell. 1990 Jun 15;61(6):1021–1033. doi: 10.1016/0092-8674(90)90067-o. [DOI] [PubMed] [Google Scholar]
  22. Johnson F. B., Parker E., Krasnow M. A. Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):739–743. doi: 10.1073/pnas.92.3.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kamps M. P., Baltimore D. E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice. Mol Cell Biol. 1993 Jan;13(1):351–357. doi: 10.1128/mcb.13.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kamps M. P., Look A. T., Baltimore D. The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials. Genes Dev. 1991 Mar;5(3):358–368. doi: 10.1101/gad.5.3.358. [DOI] [PubMed] [Google Scholar]
  25. Kamps M. P., Murre C., Sun X. H., Baltimore D. A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 1990 Feb 23;60(4):547–555. doi: 10.1016/0092-8674(90)90658-2. [DOI] [PubMed] [Google Scholar]
  26. Kamps M. P., Wright D. D. Oncoprotein E2A-Pbx1 immortalizes a myeloid progenitor in primary marrow cultures without abrogating its factor-dependence. Oncogene. 1994 Nov;9(11):3159–3166. [PubMed] [Google Scholar]
  27. 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]
  28. Knoepfler P. S., Kamps M. P. The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1. Mol Cell Biol. 1995 Oct;15(10):5811–5819. doi: 10.1128/mcb.15.10.5811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Krumlauf R. Hox genes in vertebrate development. Cell. 1994 Jul 29;78(2):191–201. doi: 10.1016/0092-8674(94)90290-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Laughon A. DNA binding specificity of homeodomains. Biochemistry. 1991 Dec 3;30(48):11357–11367. doi: 10.1021/bi00112a001. [DOI] [PubMed] [Google Scholar]
  32. Leonard J., Peers B., Johnson T., Ferreri K., Lee S., Montminy M. R. Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol. 1993 Oct;7(10):1275–1283. doi: 10.1210/mend.7.10.7505393. [DOI] [PubMed] [Google Scholar]
  33. Lu Q., Knoepfler P. S., Scheele J., Wright D. D., Kamps M. P. Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes. Mol Cell Biol. 1995 Jul;15(7):3786–3795. doi: 10.1128/mcb.15.7.3786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lu Q., Wright D. D., Kamps M. P. Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation. Mol Cell Biol. 1994 Jun;14(6):3938–3948. doi: 10.1128/mcb.14.6.3938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Monica K., Galili N., Nourse J., Saltman D., Cleary M. L. PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1. Mol Cell Biol. 1991 Dec;11(12):6149–6157. doi: 10.1128/mcb.11.12.6149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nourse J., Mellentin J. D., Galili N., Wilkinson J., Stanbridge E., Smith S. D., Cleary M. L. Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor. Cell. 1990 Feb 23;60(4):535–545. doi: 10.1016/0092-8674(90)90657-z. [DOI] [PubMed] [Google Scholar]
  41. Peers B., Sharma S., Johnson T., Kamps M., Montminy M. The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain. Mol Cell Biol. 1995 Dec;15(12):7091–7097. doi: 10.1128/mcb.15.12.7091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Pellerin I., Schnabel C., Catron K. M., Abate C. Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster. Mol Cell Biol. 1994 Jul;14(7):4532–4545. doi: 10.1128/mcb.14.7.4532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pöpperl H., Bienz M., Studer M., Chan S. K., Aparicio S., Brenner S., Mann R. S., Krumlauf R. Segmental expression of Hoxb-1 is controlled by a highly conserved autoregulatory loop dependent upon exd/pbx. Cell. 1995 Jun 30;81(7):1031–1042. doi: 10.1016/s0092-8674(05)80008-x. [DOI] [PubMed] [Google Scholar]
  45. Qian Y. Q., Billeter M., Otting G., Müller M., Gehring W. J., Wüthrich K. The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors. Cell. 1989 Nov 3;59(3):573–580. doi: 10.1016/0092-8674(89)90040-8. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Rauskolb C., Wieschaus E. Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins. EMBO J. 1994 Aug 1;13(15):3561–3569. doi: 10.1002/j.1460-2075.1994.tb06663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Saffman E. E., Krasnow M. A. A differential response element for the homeotics at the Antennapedia P1 promoter of Drosophila. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7420–7424. doi: 10.1073/pnas.91.16.7420. [DOI] [PMC free article] [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. Stark M. R., Johnson A. D. Interaction between two homeodomain proteins is specified by a short C-terminal tail. Nature. 1994 Sep 29;371(6496):429–432. doi: 10.1038/371429a0. [DOI] [PubMed] [Google Scholar]
  51. Stern S., Tanaka M., Herr W. The Oct-1 homoeodomain directs formation of a multiprotein-DNA complex with the HSV transactivator VP16. Nature. 1989 Oct 19;341(6243):624–630. doi: 10.1038/341624a0. [DOI] [PubMed] [Google Scholar]
  52. Treacy M. N., Neilson L. I., Turner E. E., He X., Rosenfeld M. G. Twin of I-POU: a two amino acid difference in the I-POU homeodomain distinguishes an activator from an inhibitor of transcription. Cell. 1992 Feb 7;68(3):491–505. doi: 10.1016/0092-8674(92)90186-g. [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. Vershon A. K., Johnson A. D. A short, disordered protein region mediates interactions between the homeodomain of the yeast alpha 2 protein and the MCM1 protein. Cell. 1993 Jan 15;72(1):105–112. doi: 10.1016/0092-8674(93)90054-t. [DOI] [PubMed] [Google Scholar]
  55. Voss J. W., Wilson L., Rosenfeld M. G. POU-domain proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can cooperate to induce expression of the prolactin promoter. Genes Dev. 1991 Jul;5(7):1309–1320. doi: 10.1101/gad.5.7.1309. [DOI] [PubMed] [Google Scholar]
  56. Wilson D., Sheng G., Lecuit T., Dostatni N., Desplan C. Cooperative dimerization of paired class homeo domains on DNA. Genes Dev. 1993 Nov;7(11):2120–2134. doi: 10.1101/gad.7.11.2120. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Xue D., Tu Y., Chalfie M. Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science. 1993 Sep 3;261(5126):1324–1328. doi: 10.1126/science.8103239. [DOI] [PubMed] [Google Scholar]
  59. Zappavigna V., Sartori D., Mavilio F. Specificity of HOX protein function depends on DNA-protein and protein-protein interactions, both mediated by the homeo domain. Genes Dev. 1994 Mar 15;8(6):732–744. doi: 10.1101/gad.8.6.732. [DOI] [PubMed] [Google Scholar]
  60. 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 Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES