Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Aug 25;23(16):3327–3334. doi: 10.1093/nar/23.16.3327

Genomic footprinting of the hsp70 and histone H3 promoters in Drosophila embryos reveals novel protein-DNA interactions.

J A Weber 1, D S Gilmour 1
PMCID: PMC307195  PMID: 7667110

Abstract

The transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress. It has been shown previously that the TBP subunit of TFIID is associated with the TATA element and that RNA polymerase II is paused downstream from the transcription start site. In order to identify new interactions involved in establishing this potentiated state, a detailed analysis of the molecular architecture of a single copy of the hsp70 promoter was performed. A suitably marked promoter was stably integrated using P-element-mediated transformation so as to overcome any ambiguity that might be associated with analyzing the five copies of the endogenous gene. Genomic footprinting using DNase I revealed two previously unidentified interactions. First, the GAGA element located at -120 is protected by protein. Secondly, the pattern of DNase I cleavage in the vicinity of the transcription start is found to bear significant similarity to the pattern associated with binding of purified TFIID. Noting that purified GAGA factor and TFIID interact similarly with the hsp70 and H3 promoters, the architecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containing a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing.

Full text

PDF
3327

Images in this article

3331Image
on p.3331
3331Image
on p.3331
3332Image
on p.3332
3333Image
on p.3333
3333Image
on p.3333

Selected References

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

  1. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  2. Cohen R. S., Meselson M. Inducible transcription and puffing in Drosophila melanogaster transformed with hsp70-phage lambda hybrid heat shock genes. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5509–5513. doi: 10.1073/pnas.81.17.5509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dudler R., Travers A. A. Upstream elements necessary for optimal function of the hsp 70 promoter in transformed flies. Cell. 1984 Sep;38(2):391–398. doi: 10.1016/0092-8674(84)90494-x. [DOI] [PubMed] [Google Scholar]
  4. Emanuel P. A., Gilmour D. S. Transcription factor TFIID recognizes DNA sequences downstream of the TATA element in the Hsp70 heat shock gene. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8449–8453. doi: 10.1073/pnas.90.18.8449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Farkas G., Gausz J., Galloni M., Reuter G., Gyurkovics H., Karch F. The Trithorax-like gene encodes the Drosophila GAGA factor. Nature. 1994 Oct 27;371(6500):806–808. doi: 10.1038/371806a0. [DOI] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  7. Garrity P. A., Wold B. J. Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1021–1025. doi: 10.1073/pnas.89.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Giardina C., Pérez-Riba M., Lis J. T. Promoter melting and TFIID complexes on Drosophila genes in vivo. Genes Dev. 1992 Nov;6(11):2190–2200. doi: 10.1101/gad.6.11.2190. [DOI] [PubMed] [Google Scholar]
  9. Gilmour D. S., Thomas G. H., Elgin S. C. Drosophila nuclear proteins bind to regions of alternating C and T residues in gene promoters. Science. 1989 Sep 29;245(4925):1487–1490. doi: 10.1126/science.2781290. [DOI] [PubMed] [Google Scholar]
  10. Glaser R. L., Thomas G. H., Siegfried E., Elgin S. C., Lis J. T. Optimal heat-induced expression of the Drosophila hsp26 gene requires a promoter sequence containing (CT)n.(GA)n repeats. J Mol Biol. 1990 Feb 20;211(4):751–761. doi: 10.1016/0022-2836(90)90075-W. [DOI] [PubMed] [Google Scholar]
  11. Grunstein M. Nucleosomes: regulators of transcription. Trends Genet. 1990 Dec;6(12):395–400. doi: 10.1016/0168-9525(90)90299-l. [DOI] [PubMed] [Google Scholar]
  12. Karpov V. L., Preobrazhenskaya O. V., Mirzabekov A. D. Chromatin structure of hsp 70 genes, activated by heat shock: selective removal of histones from the coding region and their absence from the 5' region. Cell. 1984 Feb;36(2):423–431. doi: 10.1016/0092-8674(84)90235-6. [DOI] [PubMed] [Google Scholar]
  13. Lee H., Kraus K. W., Wolfner M. F., Lis J. T. DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev. 1992 Feb;6(2):284–295. doi: 10.1101/gad.6.2.284. [DOI] [PubMed] [Google Scholar]
  14. Lis J., Wu C. Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell. 1993 Jul 16;74(1):1–4. doi: 10.1016/0092-8674(93)90286-y. [DOI] [PubMed] [Google Scholar]
  15. Lu Q., Wallrath L. L., Allan B. D., Glaser R. L., Lis J. T., Elgin S. C. Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene. J Mol Biol. 1992 Jun 20;225(4):985–998. doi: 10.1016/0022-2836(92)90099-6. [DOI] [PubMed] [Google Scholar]
  16. Lu Q., Wallrath L. L., Granok H., Elgin S. C. (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene. Mol Cell Biol. 1993 May;13(5):2802–2814. doi: 10.1128/mcb.13.5.2802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mueller P. R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. doi: 10.1126/science.2814500. [DOI] [PubMed] [Google Scholar]
  18. Nacheva G. A., Guschin D. Y., Preobrazhenskaya O. V., Karpov V. L., Ebralidse K. K., Mirzabekov A. D. Change in the pattern of histone binding to DNA upon transcriptional activation. Cell. 1989 Jul 14;58(1):27–36. doi: 10.1016/0092-8674(89)90399-1. [DOI] [PubMed] [Google Scholar]
  19. O'Brien T., Hardin S., Greenleaf A., Lis J. T. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature. 1994 Jul 7;370(6484):75–77. doi: 10.1038/370075a0. [DOI] [PubMed] [Google Scholar]
  20. Purnell B. A., Emanuel P. A., Gilmour D. S. TFIID sequence recognition of the initiator and sequences farther downstream in Drosophila class II genes. Genes Dev. 1994 Apr 1;8(7):830–842. doi: 10.1101/gad.8.7.830. [DOI] [PubMed] [Google Scholar]
  21. Rabindran S. K., Haroun R. I., Clos J., Wisniewski J., Wu C. Regulation of heat shock factor trimer formation: role of a conserved leucine zipper. Science. 1993 Jan 8;259(5092):230–234. doi: 10.1126/science.8421783. [DOI] [PubMed] [Google Scholar]
  22. Rasmussen E. B., Lis J. T. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7923–7927. doi: 10.1073/pnas.90.17.7923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rougvie A. E., Lis J. T. Postinitiation transcriptional control in Drosophila melanogaster. Mol Cell Biol. 1990 Nov;10(11):6041–6045. doi: 10.1128/mcb.10.11.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  25. Samal B., Worcel A., Louis C., Schedl P. Chromatin structure of the histone genes of D. melanogaster. Cell. 1981 Feb;23(2):401–409. doi: 10.1016/0092-8674(81)90135-5. [DOI] [PubMed] [Google Scholar]
  26. Schedl P., Artavanis-Tsakonas S., Steward R., Gehring W. J., Mirault M. E., Goldschmidt-Clermont M., Moran L., Tissières A. Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein. Cell. 1978 Aug;14(4):921–929. doi: 10.1016/0092-8674(78)90346-x. [DOI] [PubMed] [Google Scholar]
  27. Simon J. A., Sutton C. A., Lobell R. B., Glaser R. L., Lis J. T. Determinants of heat shock-induced chromosome puffing. Cell. 1985 Apr;40(4):805–817. doi: 10.1016/0092-8674(85)90340-x. [DOI] [PubMed] [Google Scholar]
  28. Sypes M. A., Gilmour D. S. Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start. Nucleic Acids Res. 1994 Mar 11;22(5):807–814. doi: 10.1093/nar/22.5.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas G. H., Elgin S. C. Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter. EMBO J. 1988 Jul;7(7):2191–2201. doi: 10.1002/j.1460-2075.1988.tb03058.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tsukiyama T., Becker P. B., Wu C. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature. 1994 Feb 10;367(6463):525–532. doi: 10.1038/367525a0. [DOI] [PubMed] [Google Scholar]
  31. Usheva A., Maldonado E., Goldring A., Lu H., Houbavi C., Reinberg D., Aloni Y. Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein. Cell. 1992 May 29;69(5):871–881. doi: 10.1016/0092-8674(92)90297-p. [DOI] [PubMed] [Google Scholar]
  32. Worcel A., Gargiulo G., Jessee B., Udvardy A., Louis C., Schedl P. Chromatin fine structure of the histone gene complex of Drosophila melanogaster. Nucleic Acids Res. 1983 Jan 25;11(2):421–439. doi: 10.1093/nar/11.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wu C. An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts. Nature. 1985 Sep 5;317(6032):84–87. doi: 10.1038/317084a0. [DOI] [PubMed] [Google Scholar]
  34. Wu C. The 5' ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature. 1980 Aug 28;286(5776):854–860. doi: 10.1038/286854a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES