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. 1991 Jul;11(7):3504–3514. doi: 10.1128/mcb.11.7.3504

Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor.

N F Cunniff 1, J Wagner 1, W D Morgan 1
PMCID: PMC361086  PMID: 1904540

Abstract

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

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Selected References

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

  1. Abravaya K., Phillips B., Morimoto R. I. Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol Cell Biol. 1991 Jan;11(1):586–592. doi: 10.1128/mcb.11.1.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amin J., Ananthan J., Voellmy R. Key features of heat shock regulatory elements. Mol Cell Biol. 1988 Sep;8(9):3761–3769. doi: 10.1128/mcb.8.9.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Bienz M., Pelham H. R. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter. Cell. 1986 Jun 6;45(5):753–760. doi: 10.1016/0092-8674(86)90789-0. [DOI] [PubMed] [Google Scholar]
  5. Bienz M., Pelham H. R. Mechanisms of heat-shock gene activation in higher eukaryotes. Adv Genet. 1987;24:31–72. doi: 10.1016/s0065-2660(08)60006-1. [DOI] [PubMed] [Google Scholar]
  6. Clos J., Westwood J. T., Becker P. B., Wilson S., Lambert K., Wu C. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell. 1990 Nov 30;63(5):1085–1097. doi: 10.1016/0092-8674(90)90511-c. [DOI] [PubMed] [Google Scholar]
  7. Cornette J. C., Evans D. B., Furlong A. M., Sharma S. K. Renin activity determination using human plasma as a substrate. Anal Biochem. 1987 May 15;163(1):93–99. doi: 10.1016/0003-2697(87)90097-2. [DOI] [PubMed] [Google Scholar]
  8. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilman M. Z., Wilson R. N., Weinberg R. A. Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression. Mol Cell Biol. 1986 Dec;6(12):4305–4316. doi: 10.1128/mcb.6.12.4305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goate A. M., Cooper D. N., Hall C., Leung T. K., Solomon E., Lim L. Localization of a human heat-shock HSP 70 gene sequence to chromosome 6 and detection of two other loci by somatic-cell hybrid and restriction fragment length polymorphism analysis. Hum Genet. 1987 Feb;75(2):123–128. doi: 10.1007/BF00591072. [DOI] [PubMed] [Google Scholar]
  11. Goldenberg C. J., Luo Y., Fenna M., Baler R., Weinmann R., Voellmy R. Purified human factor activates heat shock promoter in a HeLa cell-free transcription system. J Biol Chem. 1988 Dec 25;263(36):19734–19739. [PubMed] [Google Scholar]
  12. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  14. Greene J. M., Kingston R. E. TATA-dependent and TATA-independent function of the basal and heat shock elements of a human hsp70 promoter. Mol Cell Biol. 1990 Apr;10(4):1319–1328. doi: 10.1128/mcb.10.4.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Greene J. M., Larin Z., Taylor I. C., Prentice H., Gwinn K. A., Kingston R. E. Multiple basal elements of a human hsp70 promoter function differently in human and rodent cell lines. Mol Cell Biol. 1987 Oct;7(10):3646–3655. doi: 10.1128/mcb.7.10.3646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harrison G. S., Drabkin H. A., Kao F. T., Hartz J., Hart I. M., Chu E. H., Wu B. J., Morimoto R. I. Chromosomal location of human genes encoding major heat-shock protein HSP70. Somat Cell Mol Genet. 1987 Mar;13(2):119–130. doi: 10.1007/BF01534692. [DOI] [PubMed] [Google Scholar]
  17. Herr W. Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8009–8013. doi: 10.1073/pnas.82.23.8009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurst H. C., Jones N. C. Identification of factors that interact with the E1A-inducible adenovirus E3 promoter. Genes Dev. 1987 Dec;1(10):1132–1146. doi: 10.1101/gad.1.10.1132. [DOI] [PubMed] [Google Scholar]
  19. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  20. Kingston R. E., Schuetz T. J., Larin Z. Heat-inducible human factor that binds to a human hsp70 promoter. Mol Cell Biol. 1987 Apr;7(4):1530–1534. doi: 10.1128/mcb.7.4.1530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. Larson J. S., Schuetz T. J., Kingston R. E. Activation in vitro of sequence-specific DNA binding by a human regulatory factor. Nature. 1988 Sep 22;335(6188):372–375. doi: 10.1038/335372a0. [DOI] [PubMed] [Google Scholar]
  23. Li R., Knight J., Bream G., Stenlund A., Botchan M. Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. Genes Dev. 1989 Apr;3(4):510–526. doi: 10.1101/gad.3.4.510. [DOI] [PubMed] [Google Scholar]
  24. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  25. Liu-Johnson H. N., Gartenberg M. R., Crothers D. M. The DNA binding domain and bending angle of E. coli CAP protein. Cell. 1986 Dec 26;47(6):995–1005. doi: 10.1016/0092-8674(86)90814-7. [DOI] [PubMed] [Google Scholar]
  26. Lum L. S., Sultzman L. A., Kaufman R. J., Linzer D. I., Wu B. J. A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter. Mol Cell Biol. 1990 Dec;10(12):6709–6717. doi: 10.1128/mcb.10.12.6709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  28. Morgan W. D. Transcription factor Sp1 binds to and activates a human hsp70 gene promoter. Mol Cell Biol. 1989 Sep;9(9):4099–4104. doi: 10.1128/mcb.9.9.4099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morgan W. D., Williams G. T., Morimoto R. I., Greene J., Kingston R. E., Tjian R. Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter. Mol Cell Biol. 1987 Mar;7(3):1129–1138. doi: 10.1128/mcb.7.3.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mosser D. D., Theodorakis N. G., Morimoto R. I. Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol. 1988 Nov;8(11):4736–4744. doi: 10.1128/mcb.8.11.4736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakajima N., Horikoshi M., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol Cell Biol. 1988 Oct;8(10):4028–4040. doi: 10.1128/mcb.8.10.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Park H. O., Craig E. A. Positive and negative regulation of basal expression of a yeast HSP70 gene. Mol Cell Biol. 1989 May;9(5):2025–2033. doi: 10.1128/mcb.9.5.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perisic O., Xiao H., Lis J. T. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell. 1989 Dec 1;59(5):797–806. doi: 10.1016/0092-8674(89)90603-x. [DOI] [PubMed] [Google Scholar]
  34. Sargent C. A., Dunham I., Campbell R. D. Identification of multiple HTF-island associated genes in the human major histocompatibility complex class III region. EMBO J. 1989 Aug;8(8):2305–2312. doi: 10.1002/j.1460-2075.1989.tb08357.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sargent C. A., Dunham I., Trowsdale J., Campbell R. D. Human major histocompatibility complex contains genes for the major heat shock protein HSP70. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1968–1972. doi: 10.1073/pnas.86.6.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shuey D. J., Parker C. S. Binding of Drosophila heat-shock gene transcription factor to the hsp 70 promoter. Evidence for symmetric and dynamic interactions. J Biol Chem. 1986 Jun 15;261(17):7934–7940. [PubMed] [Google Scholar]
  37. Sorger P. K., Lewis M. J., Pelham H. R. Heat shock factor is regulated differently in yeast and HeLa cells. Nature. 1987 Sep 3;329(6134):81–84. doi: 10.1038/329081a0. [DOI] [PubMed] [Google Scholar]
  38. Sorger P. K., Nelson H. C. Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell. 1989 Dec 1;59(5):807–813. doi: 10.1016/0092-8674(89)90604-1. [DOI] [PubMed] [Google Scholar]
  39. Sorger P. K., Pelham H. R. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell. 1988 Sep 9;54(6):855–864. doi: 10.1016/s0092-8674(88)91219-6. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Taylor I. C., Solomon W., Weiner B. M., Paucha E., Bradley M., Kingston R. E. Stimulation of the human heat shock protein 70 promoter in vitro by simian virus 40 large T antigen. J Biol Chem. 1989 Sep 25;264(27):16160–16164. [PubMed] [Google Scholar]
  42. Theodorakis N. G., Zand D. J., Kotzbauer P. T., Williams G. T., Morimoto R. I. Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response. Mol Cell Biol. 1989 Aug;9(8):3166–3173. doi: 10.1128/mcb.9.8.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tullius T. D., Dombroski B. A., Churchill M. E., Kam L. Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts. Methods Enzymol. 1987;155:537–558. doi: 10.1016/0076-6879(87)55035-2. [DOI] [PubMed] [Google Scholar]
  44. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Varshavsky A. Electrophoretic assay for DNA-binding proteins. Methods Enzymol. 1987;151:551–565. doi: 10.1016/s0076-6879(87)51044-8. [DOI] [PubMed] [Google Scholar]
  46. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  47. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  48. Wiederrecht G., Seto D., Parker C. S. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell. 1988 Sep 9;54(6):841–853. doi: 10.1016/s0092-8674(88)91197-x. [DOI] [PubMed] [Google Scholar]
  49. Williams G. T., McClanahan T. K., Morimoto R. I. E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. Mol Cell Biol. 1989 Jun;9(6):2574–2587. doi: 10.1128/mcb.9.6.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wu B. J., Hurst H. C., Jones N. C., Morimoto R. I. The E1A 13S product of adenovirus 5 activates transcription of the cellular human HSP70 gene. Mol Cell Biol. 1986 Aug;6(8):2994–2999. doi: 10.1128/mcb.6.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wu B. J., Kingston R. E., Morimoto R. I. Human HSP70 promoter contains at least two distinct regulatory domains. Proc Natl Acad Sci U S A. 1986 Feb;83(3):629–633. doi: 10.1073/pnas.83.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wu C., Wilson S., Walker B., Dawid I., Paisley T., Zimarino V., Ueda H. Purification and properties of Drosophila heat shock activator protein. Science. 1987 Nov 27;238(4831):1247–1253. doi: 10.1126/science.3685975. [DOI] [PubMed] [Google Scholar]
  53. Xiao H., Lis J. T. Germline transformation used to define key features of heat-shock response elements. Science. 1988 Mar 4;239(4844):1139–1142. doi: 10.1126/science.3125608. [DOI] [PubMed] [Google Scholar]
  54. Zimarino V., Tsai C., Wu C. Complex modes of heat shock factor activation. Mol Cell Biol. 1990 Feb;10(2):752–759. doi: 10.1128/mcb.10.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zimarino V., Wu C. Induction of sequence-specific binding of Drosophila heat shock activator protein without protein synthesis. 1987 Jun 25-Jul 1Nature. 327(6124):727–730. doi: 10.1038/327727a0. [DOI] [PubMed] [Google Scholar]
  56. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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