A comparison of in vivo and in vitro DNA-binding specificities suggests a new model for homeoprotein DNA binding in Drosophila embryos (original) (raw)

Abstract

Little is known about the range of DNA sequences bound by transcription factors in vivo. Using a sensitive UV cross-linking technique, we show that three classes of homeoprotein bind at significant levels to the majority of genes in Drosophila embryos. The three classes bind with specificities different from each other; however, their levels of binding on any single DNA fragment differ by no more than 5- to 10-fold. On actively transcribed genes, there is a good correlation between the in vivo DNA-binding specificity of each class and its in vitro DNA-binding specificity. In contrast, no such correlation is seen on inactive or weakly transcribed genes. These genes are bound poorly in vivo, even though they contain many high affinity homeoprotein-binding sites. Based on these results, we suggest how the in vivo pattern of homeoprotein DNA binding is determined.

Full Text

The Full Text of this article is available as a PDF (553.5 KB).

Selected References

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

  1. Akam M. E., Martinez-Arias A. The distribution of Ultrabithorax transcripts in Drosophila embryos. EMBO J. 1985 Jul;4(7):1689–1700. doi: 10.1002/j.1460-2075.1985.tb03838.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beato M., Eisfeld K. Transcription factor access to chromatin. Nucleic Acids Res. 1997 Sep 15;25(18):3559–3563. doi: 10.1093/nar/25.18.3559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biggin M. D., McGinnis W. Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity. Development. 1997 Nov;124(22):4425–4433. doi: 10.1242/dev.124.22.4425. [DOI] [PubMed] [Google Scholar]
  4. Blatter E. E., Ebright Y. W., Ebright R. H. Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking. Nature. 1992 Oct 15;359(6396):650–652. doi: 10.1038/359650a0. [DOI] [PubMed] [Google Scholar]
  5. Boyd K. E., Wells J., Gutman J., Bartley S. M., Farnham P. J. c-Myc target gene specificity is determined by a post-DNAbinding mechanism. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13887–13892. doi: 10.1073/pnas.95.23.13887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Driever W., Nüsslein-Volhard C. A gradient of bicoid protein in Drosophila embryos. Cell. 1988 Jul 1;54(1):83–93. doi: 10.1016/0092-8674(88)90182-1. [DOI] [PubMed] [Google Scholar]
  7. Driever W., Nüsslein-Volhard C. The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo. Nature. 1989 Jan 12;337(6203):138–143. doi: 10.1038/337138a0. [DOI] [PubMed] [Google Scholar]
  8. Faisst S., Meyer S. Compilation of vertebrate-encoded transcription factors. Nucleic Acids Res. 1992 Jan 11;20(1):3–26. doi: 10.1093/nar/20.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujioka M., Miskiewicz P., Raj L., Gulledge A. A., Weir M., Goto T. Drosophila Paired regulates late even-skipped expression through a composite binding site for the paired domain and the homeodomain. Development. 1996 Sep;122(9):2697–2707. doi: 10.1242/dev.122.9.2697. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Guichet A., Copeland J. W., Erdélyi M., Hlousek D., Závorszky P., Ho J., Brown S., Percival-Smith A., Krause H. M., Ephrussi A. The nuclear receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually dependent cofactors. Nature. 1997 Feb 6;385(6616):548–552. doi: 10.1038/385548a0. [DOI] [PubMed] [Google Scholar]
  12. Gutjahr T., Frei E., Noll M. Complex regulation of early paired expression: initial activation by gap genes and pattern modulation by pair-rule genes. Development. 1993 Feb;117(2):609–623. doi: 10.1242/dev.117.2.609. [DOI] [PubMed] [Google Scholar]
  13. Hanes S. D., Riddihough G., Ish-Horowicz D., Brent R. Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen. Mol Cell Biol. 1994 May;14(5):3364–3375. doi: 10.1128/mcb.14.5.3364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heinemeyer T., Wingender E., Reuter I., Hermjakob H., Kel A. E., Kel O. V., Ignatieva E. V., Ananko E. A., Podkolodnaya O. A., Kolpakov F. A. Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res. 1998 Jan 1;26(1):362–367. doi: 10.1093/nar/26.1.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Hockensmith J. W., Kubasek W. L., Vorachek W. R., Evertsz E. M., von Hippel P. H. Laser cross-linking of protein-nucleic acid complexes. Methods Enzymol. 1991;208:211–236. doi: 10.1016/0076-6879(91)08015-a. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Jiang J., Hoey T., Levine M. Autoregulation of a segmentation gene in Drosophila: combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element. Genes Dev. 1991 Feb;5(2):265–277. doi: 10.1101/gad.5.2.265. [DOI] [PubMed] [Google Scholar]
  19. Jun S., Desplan C. Cooperative interactions between paired domain and homeodomain. Development. 1996 Sep;122(9):2639–2650. doi: 10.1242/dev.122.9.2639. [DOI] [PubMed] [Google Scholar]
  20. Kadonaga J. T. Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines. Cell. 1998 Feb 6;92(3):307–313. doi: 10.1016/s0092-8674(00)80924-1. [DOI] [PubMed] [Google Scholar]
  21. Laney J. D., Biggin M. D. Zeste-mediated activation by an enhancer is independent of cooperative DNA binding in vivo. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3602–3604. doi: 10.1073/pnas.94.8.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liang Z., Biggin M. D. Eve and ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila. Development. 1998 Nov;125(22):4471–4482. doi: 10.1242/dev.125.22.4471. [DOI] [PubMed] [Google Scholar]
  23. Lin S., Riggs A. D. The general affinity of lac repressor for E. coli DNA: implications for gene regulation in procaryotes and eucaryotes. Cell. 1975 Feb;4(2):107–111. doi: 10.1016/0092-8674(75)90116-6. [DOI] [PubMed] [Google Scholar]
  24. Mann R. S., Chan S. K. Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins. Trends Genet. 1996 Jul;12(7):258–262. doi: 10.1016/0168-9525(96)10026-3. [DOI] [PubMed] [Google Scholar]
  25. Margolis J. S., Borowsky M. L., Steingrímsson E., Shim C. W., Lengyel J. A., Posakony J. W. Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element. Development. 1995 Sep;121(9):3067–3077. doi: 10.1242/dev.121.9.3067. [DOI] [PubMed] [Google Scholar]
  26. O'Brien T., Wilkins R. C., Giardina C., Lis J. T. Distribution of GAGA protein on Drosophila genes in vivo. Genes Dev. 1995 May 1;9(9):1098–1110. doi: 10.1101/gad.9.9.1098. [DOI] [PubMed] [Google Scholar]
  27. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Ptashne M., Gann A. Transcriptional activation by recruitment. Nature. 1997 Apr 10;386(6625):569–577. doi: 10.1038/386569a0. [DOI] [PubMed] [Google Scholar]
  30. Sauer F., Hansen S. K., Tjian R. Multiple TAFIIs directing synergistic activation of transcription. Science. 1995 Dec 15;270(5243):1783–1788. doi: 10.1126/science.270.5243.1783. [DOI] [PubMed] [Google Scholar]
  31. Simpson-Brose M., Treisman J., Desplan C. Synergy between the hunchback and bicoid morphogens is required for anterior patterning in Drosophila. Cell. 1994 Sep 9;78(5):855–865. doi: 10.1016/s0092-8674(94)90622-x. [DOI] [PubMed] [Google Scholar]
  32. Small S., Blair A., Levine M. Regulation of even-skipped stripe 2 in the Drosophila embryo. EMBO J. 1992 Nov;11(11):4047–4057. doi: 10.1002/j.1460-2075.1992.tb05498.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. TenHarmsel A., Austin R. J., Savenelli N., Biggin M. D. Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing. Mol Cell Biol. 1993 May;13(5):2742–2752. doi: 10.1128/mcb.13.5.2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Urness L. D., Thummel C. S. Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein. Cell. 1990 Oct 5;63(1):47–61. doi: 10.1016/0092-8674(90)90287-o. [DOI] [PubMed] [Google Scholar]
  36. Wallrath L. L., Lu Q., Granok H., Elgin S. C. Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures. Bioessays. 1994 Mar;16(3):165–170. doi: 10.1002/bies.950160306. [DOI] [PubMed] [Google Scholar]
  37. Walter J., Biggin M. D. DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2680–2685. doi: 10.1073/pnas.93.7.2680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Walter J., Biggin M. D. Measurement of in vivo DNA binding by sequence-specific transcription factors using UV cross-linking. Methods. 1997 Feb;11(2):215–224. doi: 10.1006/meth.1996.0408. [DOI] [PubMed] [Google Scholar]
  39. Walter J., Dever C. A., Biggin M. D. Two homeo domain proteins bind with similar specificity to a wide range of DNA sites in Drosophila embryos. Genes Dev. 1994 Jul 15;8(14):1678–1692. doi: 10.1101/gad.8.14.1678. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wu C. Two protein-binding sites in chromatin implicated in the activation of heat-shock genes. Nature. 1984 May 17;309(5965):229–234. doi: 10.1038/309229a0. [DOI] [PubMed] [Google Scholar]
  42. Yang S. W., Nash H. A. Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target. EMBO J. 1995 Dec 15;14(24):6292–6300. doi: 10.1002/j.1460-2075.1995.tb00319.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yao T. P., Forman B. M., Jiang Z., Cherbas L., Chen J. D., McKeown M., Cherbas P., Evans R. M. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature. 1993 Dec 2;366(6454):476–479. doi: 10.1038/366476a0. [DOI] [PubMed] [Google Scholar]
  44. Yu Y., Li W., Su K., Yussa M., Han W., Perrimon N., Pick L. The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz. Nature. 1997 Feb 6;385(6616):552–555. doi: 10.1038/385552a0. [DOI] [PubMed] [Google Scholar]
  45. von Hippel P. H., Revzin A., Gross C. A., Wang A. C. Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: equilibrium aspects. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4808–4812. doi: 10.1073/pnas.71.12.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]