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. 1997 Dec 1;25(23):4730–4739. doi: 10.1093/nar/25.23.4730

Homeodomain-DNA interactions of the Pho2 protein are promoter-dependent.

M C Justice 1, B P Hogan 1, A K Vershon 1
PMCID: PMC147108  PMID: 9365251

Abstract

The homeodomain (HD) is a conserved sequence-specific DNA-binding motif found in many eukaryotic transcriptional regulatory proteins. Despite the wealth of in vitro data on the mechanism HD proteins use to bind DNA, comparatively little is known about the roles of individual residues in these domains in vivo . The Saccharomyces cerevisiae Pho2 protein contains a HD that shares significant sequence identity with the Drosophila Engrailed protein. We have used the co-crystal structure of Engrailed as a model to predict how Pho2 might contact DNA and have examined how individual residues of the Pho2 HD contribute to transcriptional activation in vivo and to DNA binding in vitro. Our results demonstrate that Pho2 and Engrailed share many similar DNA-binding characteristics. However, our results also show that some highly conserved residues, which contact the DNA in many HD structures, make relatively small contributions to the DNA-binding affinity and in vivo activity of the Pho2 protein. We also show that the N-terminal arm of the Pho2 HD is a critical component in determining the DNA-binding specificity of the protein and that the requirements for residues in the N-terminal arm are promoter-dependent for Pho2 transcriptional activation and DNA binding.

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Selected References

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  1. Ades S. E., Sauer R. T. Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50. Biochemistry. 1994 Aug 9;33(31):9187–9194. doi: 10.1021/bi00197a022. [DOI] [PubMed] [Google Scholar]
  2. Ades S. E., Sauer R. T. Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex. Biochemistry. 1995 Nov 7;34(44):14601–14608. doi: 10.1021/bi00044a040. [DOI] [PubMed] [Google Scholar]
  3. Arndt K. T., Styles C., Fink G. R. Multiple global regulators control HIS4 transcription in yeast. Science. 1987 Aug 21;237(4817):874–880. doi: 10.1126/science.3303332. [DOI] [PubMed] [Google Scholar]
  4. Barbarić S., Münsterkötter M., Svaren J., Hörz W. The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter. Nucleic Acids Res. 1996 Nov 15;24(22):4479–4486. doi: 10.1093/nar/24.22.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Billeter M., Qian Y. Q., Otting G., Müller M., Gehring W., Wüthrich K. Determination of the nuclear magnetic resonance solution structure of an Antennapedia homeodomain-DNA complex. J Mol Biol. 1993 Dec 20;234(4):1084–1093. doi: 10.1006/jmbi.1993.1661. [DOI] [PubMed] [Google Scholar]
  6. Bostian K. A., Lemire J. M., Cannon L. E., Halvorson H. O. In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4504–4508. doi: 10.1073/pnas.77.8.4504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braus G., Mösch H. U., Vogel K., Hinnen A., Hütter R. Interpathway regulation of the TRP4 gene of yeast. EMBO J. 1989 Mar;8(3):939–945. doi: 10.1002/j.1460-2075.1989.tb03455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brazas R. M., Stillman D. J. Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein. Mol Cell Biol. 1993 Sep;13(9):5524–5537. doi: 10.1128/mcb.13.9.5524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brazas R. M., Stillman D. J. The Swi5 zinc-finger and Grf10 homeodomain proteins bind DNA cooperatively at the yeast HO promoter. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11237–11241. doi: 10.1073/pnas.90.23.11237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bürglin T. R. The yeast regulatory gene PHO2 encodes a homeo box. Cell. 1988 May 6;53(3):339–340. doi: 10.1016/0092-8674(88)90153-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Daignan-Fornier B., Fink G. R. Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6746–6750. doi: 10.1073/pnas.89.15.6746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devlin C., Tice-Baldwin K., Shore D., Arndt K. T. RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene. Mol Cell Biol. 1991 Jul;11(7):3642–3651. doi: 10.1128/mcb.11.7.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ekker S. C., Jackson D. G., von Kessler D. P., Sun B. I., Young K. E., Beachy P. A. The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J. 1994 Aug 1;13(15):3551–3560. doi: 10.1002/j.1460-2075.1994.tb06662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  19. Goutte C., Johnson A. D. a1 protein alters the DNA binding specificity of alpha 2 repressor. Cell. 1988 Mar 25;52(6):875–882. doi: 10.1016/0092-8674(88)90429-1. [DOI] [PubMed] [Google Scholar]
  20. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  21. Hanes S. D., Brent R. DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9. Cell. 1989 Jun 30;57(7):1275–1283. doi: 10.1016/0092-8674(89)90063-9. [DOI] [PubMed] [Google Scholar]
  22. Hirsch J. A., Aggarwal A. K. Structure of the even-skipped homeodomain complexed to AT-rich DNA: new perspectives on homeodomain specificity. EMBO J. 1995 Dec 15;14(24):6280–6291. doi: 10.1002/j.1460-2075.1995.tb00318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirst K., Fisher F., McAndrew P. C., Goding C. R. The transcription factor, the Cdk, its cyclin and their regulator: directing the transcriptional response to a nutritional signal. EMBO J. 1994 Nov 15;13(22):5410–5420. doi: 10.1002/j.1460-2075.1994.tb06876.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Keleher C. A., Goutte C., Johnson A. D. The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein. Cell. 1988 Jun 17;53(6):927–936. doi: 10.1016/s0092-8674(88)90449-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Klemm J. D., Rould M. A., Aurora R., Herr W., Pabo C. O. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell. 1994 Apr 8;77(1):21–32. doi: 10.1016/0092-8674(94)90231-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Li T., Stark M. R., Johnson A. D., Wolberger C. Crystal structure of the MATa1/MAT alpha 2 homeodomain heterodimer bound to DNA. Science. 1995 Oct 13;270(5234):262–269. doi: 10.1126/science.270.5234.262. [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. 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]
  32. 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]
  33. Mead J., Zhong H., Acton T. B., Vershon A. K. The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes. Mol Cell Biol. 1996 May;16(5):2135–2143. doi: 10.1128/mcb.16.5.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. O'Connell K. F., Baker R. E. Possible cross-regulation of phosphate and sulfate metabolism in Saccharomyces cerevisiae. Genetics. 1992 Sep;132(1):63–73. doi: 10.1093/genetics/132.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Otting G., Qian Y. Q., Billeter M., Müller M., Affolter M., Gehring W. J., Wüthrich K. Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution. EMBO J. 1990 Oct;9(10):3085–3092. doi: 10.1002/j.1460-2075.1990.tb07505.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Phelan M. L., Featherstone M. S. Distinct HOX N-terminal arm residues are responsible for specificity of DNA recognition by HOX monomers and HOX.PBX heterodimers. J Biol Chem. 1997 Mar 28;272(13):8635–8643. doi: 10.1074/jbc.272.13.8635. [DOI] [PubMed] [Google Scholar]
  37. Phelan M. L., Sadoul R., Featherstone M. S. Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm. Mol Cell Biol. 1994 Aug;14(8):5066–5075. doi: 10.1128/mcb.14.8.5066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prodromou C., Pearl L. H. Recursive PCR: a novel technique for total gene synthesis. Protein Eng. 1992 Dec;5(8):827–829. doi: 10.1093/protein/5.8.827. [DOI] [PubMed] [Google Scholar]
  39. Sengstag C., Hinnen A. A 28-bp segment of the Saccharomyces cerevisiae PHO5 upstream activator sequence confers phosphate control to the CYC1-lacZ gene fusion. Gene. 1988 Jul 30;67(2):223–228. doi: 10.1016/0378-1119(88)90399-x. [DOI] [PubMed] [Google Scholar]
  40. Shang Z., Isaac V. E., Li H., Patel L., Catron K. M., Curran T., Montelione G. T., Abate C. Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8373–8377. doi: 10.1073/pnas.91.18.8373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tice-Baldwin K., Fink G. R., Arndt K. T. BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2. Science. 1989 Nov 17;246(4932):931–935. doi: 10.1126/science.2683089. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Vershon A. K., Jin Y., Johnson A. D. A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression. Genes Dev. 1995 Jan 15;9(2):182–192. doi: 10.1101/gad.9.2.182. [DOI] [PubMed] [Google Scholar]
  44. Vogel K., Hörz W., Hinnen A. The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions. Mol Cell Biol. 1989 May;9(5):2050–2057. doi: 10.1128/mcb.9.5.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wilson D. S., Guenther B., Desplan C., Kuriyan J. High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA. Cell. 1995 Sep 8;82(5):709–719. doi: 10.1016/0092-8674(95)90468-9. [DOI] [PubMed] [Google Scholar]
  46. Wilson D. S., Sheng G., Jun S., Desplan C. Conservation and diversification in homeodomain-DNA interactions: a comparative genetic analysis. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6886–6891. doi: 10.1073/pnas.93.14.6886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Zeng W., Andrew D. J., Mathies L. D., Horner M. A., Scott M. P. Ectopic expression and function of the Antp and Scr homeotic genes: the N terminus of the homeodomain is critical to functional specificity. Development. 1993 Jun;118(2):339–352. doi: 10.1242/dev.118.2.339. [DOI] [PubMed] [Google Scholar]
  49. Zhang F., Kirouac M., Zhu N., Hinnebusch A. G., Rolfes R. J. Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription. Mol Cell Biol. 1997 Jun;17(6):3272–3283. doi: 10.1128/mcb.17.6.3272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]

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