Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jul 15;25(14):2847–2853. doi: 10.1093/nar/25.14.2847

Cysteine 50 of the POU H domain determines the range of targets recognized by POU proteins.

A G Stepchenko 1, N N Luchina 1, E V Pankratova 1
PMCID: PMC146835  PMID: 9207034

Abstract

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT. POU proteins also recognize, with weaker affinity, the TAAT-like targets of another group of regulatory factors, the homeoproteins. Up to now, it has not been known why Cys50 of the POUHdomain is absolutely conserved in contrast to that in homeoproteins. To assess the importance of Cys50 in determining the binding specificity of POU proteins, all possible amino acids were substituted for Cys at position 50, and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homeospecific binding sites. Only the wild-type POU was shown to adequately discriminate between the octamer and homeospecific sites, and the protein affinity was only slightly affected by the nucleotide sequence flanking the octamer at the 3'-end. Any amino acid substitution at position 50 resulted in the mutant protein binding efficiently both to the octamer and the TAAT-like sequences. Moreover, in this case the 3'-flanking sequences influenced the binding to a much greater extent.

Full Text

The Full Text of this article is available as a PDF (310.7 KB).

Selected References

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

  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. Banerji J., Olson L., Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. doi: 10.1016/0092-8674(83)90015-6. [DOI] [PubMed] [Google Scholar]
  3. Baumruker T., Sturm R., Herr W. OBP100 binds remarkably degenerate octamer motifs through specific interactions with flanking sequences. Genes Dev. 1988 Nov;2(11):1400–1413. doi: 10.1101/gad.2.11.1400. [DOI] [PubMed] [Google Scholar]
  4. Botfield M. C., Jancso A., Weiss M. A. An invariant asparagine in the POU-specific homeodomain regulates the specificity of the Oct-2 POU motif. Biochemistry. 1994 Jul 5;33(26):8113–8121. doi: 10.1021/bi00192a016. [DOI] [PubMed] [Google Scholar]
  5. Brugnera E., Xu L., Schaffner W., Arnosti D. N. POU-specific domain of Oct-2 factor confers 'octamer' motif DNA binding specificity on heterologous Antennapedia homeodomain. FEBS Lett. 1992 Dec 21;314(3):361–365. doi: 10.1016/0014-5793(92)81506-h. [DOI] [PubMed] [Google Scholar]
  6. Clerc R. G., Corcoran L. M., LeBowitz J. H., Baltimore D., Sharp P. A. The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains. Genes Dev. 1988 Dec;2(12A):1570–1581. doi: 10.1101/gad.2.12a.1570. [DOI] [PubMed] [Google Scholar]
  7. Douville P., Hagmann M., Georgiev O., Schaffner W. Positive and negative regulation at the herpes simplex virus ICP4 and ICP0 TAATGARAT motifs. Virology. 1995 Feb 20;207(1):107–116. doi: 10.1006/viro.1995.1056. [DOI] [PubMed] [Google Scholar]
  8. Falkner F. G., Zachau H. G. Correct transcription of an immunoglobulin kappa gene requires an upstream fragment containing conserved sequence elements. Nature. 1984 Jul 5;310(5972):71–74. doi: 10.1038/310071a0. [DOI] [PubMed] [Google Scholar]
  9. Gstaiger M., Georgiev O., van Leeuwen H., van der Vliet P., Schaffner W. The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation. EMBO J. 1996 Jun 3;15(11):2781–2790. [PMC free article] [PubMed] [Google Scholar]
  10. Gstaiger M., Knoepfel L., Georgiev O., Schaffner W., Hovens C. M. A B-cell coactivator of octamer-binding transcription factors. Nature. 1995 Jan 26;373(6512):360–362. doi: 10.1038/373360a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Lebowitz P., Ghosh P. K. Initiation and regulation of simian virus 40 early transcription in vitro. J Virol. 1982 Feb;41(2):449–461. doi: 10.1128/jvi.41.2.449-461.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Luo Y., Roeder R. G. Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B. Mol Cell Biol. 1995 Aug;15(8):4115–4124. doi: 10.1128/mcb.15.8.4115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  19. O'Hare P., Goding C. R. Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell. 1988 Feb 12;52(3):435–445. doi: 10.1016/s0092-8674(88)80036-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Pomerantz J. L., Sharp P. A. Homeodomain determinants of major groove recognition. Biochemistry. 1994 Sep 13;33(36):10851–10858. doi: 10.1021/bi00202a001. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ruvkun G., Finney M. Regulation of transcription and cell identity by POU domain proteins. Cell. 1991 Feb 8;64(3):475–478. doi: 10.1016/0092-8674(91)90227-p. [DOI] [PubMed] [Google Scholar]
  24. Schubart D. B., Sauter P., Massa S., Friedl E. M., Schwarzenbach H., Matthias P. Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1. Nucleic Acids Res. 1996 May 15;24(10):1913–1920. doi: 10.1093/nar/24.10.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stepchenko A. G. Noncanonical Oct-sequences are targets for mouse Oct-2B transcription factor. FEBS Lett. 1994 Jan 10;337(2):175–178. doi: 10.1016/0014-5793(94)80268-8. [DOI] [PubMed] [Google Scholar]
  28. Stepchenko A. G. Vzaimodeistvie Oct-sviazyvaiushchikh faktorov transkriptsii s bol'shim naborom "nekanonicheskikh" oct-posledovatel'nostei. Pervichnaia posledovatel'nost' Oct-2B kDNK myshi. Dokl Akad Nauk. 1992;325(1):175–178. [PubMed] [Google Scholar]
  29. Strubin M., Newell J. W., Matthias P. OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. Cell. 1995 Feb 10;80(3):497–506. doi: 10.1016/0092-8674(95)90500-6. [DOI] [PubMed] [Google Scholar]
  30. Sturm R., Baumruker T., Franza B. R., Jr, Herr W. A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer. Genes Dev. 1987 Dec;1(10):1147–1160. doi: 10.1101/gad.1.10.1147. [DOI] [PubMed] [Google Scholar]
  31. Topol J., Ruden D. M., Parker C. S. Sequences required for in vitro transcriptional activation of a Drosophila hsp 70 gene. Cell. 1985 Sep;42(2):527–537. doi: 10.1016/0092-8674(85)90110-2. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Verrijzer C. P., Alkema M. J., van Weperen W. W., Van Leeuwen H. C., Strating M. J., van der Vliet P. C. The DNA binding specificity of the bipartite POU domain and its subdomains. EMBO J. 1992 Dec;11(13):4993–5003. doi: 10.1002/j.1460-2075.1992.tb05606.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Verrijzer C. P., Kal A. J., van der Vliet P. C. The oct-1 homeo domain contacts only part of the octamer sequence and full oct-1 DNA-binding activity requires the POU-specific domain. Genes Dev. 1990 Nov;4(11):1964–1974. doi: 10.1101/gad.4.11.1964. [DOI] [PubMed] [Google Scholar]
  35. Verrijzer C. P., Van der Vliet P. C. POU domain transcription factors. Biochim Biophys Acta. 1993 Apr 29;1173(1):1–21. doi: 10.1016/0167-4781(93)90237-8. [DOI] [PubMed] [Google Scholar]
  36. Verrijzer C. P., van Oosterhout J. A., van Weperen W. W., van der Vliet P. C. POU proteins bend DNA via the POU-specific domain. EMBO J. 1991 Oct;10(10):3007–3014. doi: 10.1002/j.1460-2075.1991.tb07851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wey E., Schäfer B. W. Identification of novel DNA binding sites recognized by the transcription factor mPOU (POU6F1). Biochem Biophys Res Commun. 1996 Mar 18;220(2):274–279. doi: 10.1006/bbrc.1996.0395. [DOI] [PubMed] [Google Scholar]
  38. apRhys C. M., Ciufo D. M., O'Neill E. A., Kelly T. J., Hayward G. S. Overlapping octamer and TAATGARAT motifs in the VF65-response elements in herpes simplex virus immediate-early promoters represent independent binding sites for cellular nuclear factor III. J Virol. 1989 Jun;63(6):2798–2812. doi: 10.1128/jvi.63.6.2798-2812.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES