Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Nov 15;25(22):4537–4544. doi: 10.1093/nar/25.22.4537

High affinity binding of MEF-2C correlates with DNA bending.

D Meierhans 1, M Sieber 1, R K Allemann 1
PMCID: PMC147066  PMID: 9358163

Abstract

To regulate lineage-specific gene expression in many cell types, members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors cooperate with basic helix-loop-helix (bHLH) proteins, which show only limited intrinsic DNA binding specificity. We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural characteristics recognized by MEF-2C. Measurements of the apparent dissociation constants of MEF-2C complexes with several DNA sequences revealed that MEF-2C bound with high affinity to DNA sequences containing a MEF site. Mutations in the MEF site which did not affect the bendability of the DNA changed the free energy of binding only marginally. However, reducing the intrinsic bendability of the DNA binding site through an AA-->GC substitution increased the half-maximal binding concentration of MEF-2C by almost one order of magnitude. Electrophoretic mobility shift assays revealed markedly reduced MEF-2C binding to DNA containing 2,6-diaminopurine. On binding to MEF-2C the maximum ellipticity at 275 nm in the CD spectrum of DNA containing a MEF site was red shifted by 4 nm and its intensity reduced significantly, while a slight blue shift of <1 nm was observed for a mutant DNA sequence with reduced bendability (AA-->GC). Bending analysis by circular permutation assay revealed that the DNA in the cognate complex was bent by 49 degrees , while the DNA in the complex with the mutant oligonucleotide was largely unbent.

Full Text

The Full Text of this article is available as a PDF (446.6 KB).

Selected References

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

  1. Allemann R. K., Egli M. DNA recognition and bending. Chem Biol. 1997 Sep;4(9):643–650. doi: 10.1016/s1074-5521(97)90218-0. [DOI] [PubMed] [Google Scholar]
  2. Bailly C., Waring M. J., Travers A. A. Effects of base substitutions on the binding of a DNA-bending protein. J Mol Biol. 1995 Oct 13;253(1):1–7. doi: 10.1006/jmbi.1995.0530. [DOI] [PubMed] [Google Scholar]
  3. Berger C., Jelesarov I., Bosshard H. R. Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry. Biochemistry. 1996 Nov 26;35(47):14984–14991. doi: 10.1021/bi961312a. [DOI] [PubMed] [Google Scholar]
  4. Blazy B., Culard F., Maurizot J. C. Interaction between the cyclic AMP receptor protein and DNA. Conformational studies. J Mol Biol. 1987 May 5;195(1):175–183. doi: 10.1016/0022-2836(87)90334-2. [DOI] [PubMed] [Google Scholar]
  5. Breitbart R. E., Liang C. S., Smoot L. B., Laheru D. A., Mahdavi V., Nadal-Ginard B. A fourth human MEF2 transcription factor, hMEF2D, is an early marker of the myogenic lineage. Development. 1993 Aug;118(4):1095–1106. doi: 10.1242/dev.118.4.1095. [DOI] [PubMed] [Google Scholar]
  6. Brukner I., Sánchez R., Suck D., Pongor S. Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides. EMBO J. 1995 Apr 18;14(8):1812–1818. doi: 10.1002/j.1460-2075.1995.tb07169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buckingham M. Making muscle in mammals. Trends Genet. 1992 Apr;8(4):144–148. doi: 10.1016/0168-9525(92)90373-C. [DOI] [PubMed] [Google Scholar]
  8. Cheng T. C., Wallace M. C., Merlie J. P., Olson E. N. Separable regulatory elements governing myogenin transcription in mouse embryogenesis. Science. 1993 Jul 9;261(5118):215–218. doi: 10.1126/science.8392225. [DOI] [PubMed] [Google Scholar]
  9. Connor F., Cary P. D., Read C. M., Preston N. S., Driscoll P. C., Denny P., Crane-Robinson C., Ashworth A. DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5. Nucleic Acids Res. 1994 Aug 25;22(16):3339–3346. doi: 10.1093/nar/22.16.3339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dickerson R. E., Drew H. R. Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure. J Mol Biol. 1981 Jul 15;149(4):761–786. doi: 10.1016/0022-2836(81)90357-0. [DOI] [PubMed] [Google Scholar]
  11. Dickerson R. E., Goodsell D. S., Neidle S. "...the tyranny of the lattice...". Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3579–3583. doi: 10.1073/pnas.91.9.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dickerson R. E., Goodsell D., Kopka M. L. MPD and DNA bending in crystals and in solution. J Mol Biol. 1996 Feb 16;256(1):108–125. doi: 10.1006/jmbi.1996.0071. [DOI] [PubMed] [Google Scholar]
  13. Diekmann S., von Kitzing E., McLaughlin L., Ott J., Eckstein F. The influence of exocyclic substituents of purine bases on DNA curvature. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8257–8261. doi: 10.1073/pnas.84.23.8257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Drew H. R., Samson S., Dickerson R. E. Structure of a B-DNA dodecamer at 16 K. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4040–4044. doi: 10.1073/pnas.79.13.4040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emerson C. P., Jr Skeletal myogenesis: genetics and embryology to the fore. Curr Opin Genet Dev. 1993 Apr;3(2):265–274. doi: 10.1016/0959-437x(93)90033-l. [DOI] [PubMed] [Google Scholar]
  16. Ferrari S., Harley V. R., Pontiggia A., Goodfellow P. N., Lovell-Badge R., Bianchi M. E. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992 Dec;11(12):4497–4506. doi: 10.1002/j.1460-2075.1992.tb05551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ferré-D'Amaré A. R., Prendergast G. C., Ziff E. B., Burley S. K. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature. 1993 May 6;363(6424):38–45. doi: 10.1038/363038a0. [DOI] [PubMed] [Google Scholar]
  18. Gabrielian A., Pongor S. Correlation of intrinsic DNA curvature with DNA property periodicity. FEBS Lett. 1996 Sep 9;393(1):65–68. doi: 10.1016/0014-5793(96)00855-1. [DOI] [PubMed] [Google Scholar]
  19. Gabrielian A., Simoncsits A., Pongor S. Distribution of bending propensity in DNA sequences. FEBS Lett. 1996 Sep 9;393(1):124–130. doi: 10.1016/0014-5793(96)00837-x. [DOI] [PubMed] [Google Scholar]
  20. Garcia R. A., Bustamante C. J., Reich N. O. Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7618–7622. doi: 10.1073/pnas.93.15.7618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Glover J. N., Harrison S. C. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature. 1995 Jan 19;373(6511):257–261. doi: 10.1038/373257a0. [DOI] [PubMed] [Google Scholar]
  22. Gossett L. A., Kelvin D. J., Sternberg E. A., Olson E. N. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol. 1989 Nov;9(11):5022–5033. doi: 10.1128/mcb.9.11.5022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Greenfield N., Fasman G. D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969 Oct;8(10):4108–4116. doi: 10.1021/bi00838a031. [DOI] [PubMed] [Google Scholar]
  24. Ivanov V. I., Minchenkova L. E., Schyolkina A. K., Poletayev A. I. Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism. Biopolymers. 1973;12(1):89–110. doi: 10.1002/bip.1973.360120109. [DOI] [PubMed] [Google Scholar]
  25. Juo Z. S., Chiu T. K., Leiberman P. M., Baikalov I., Berk A. J., Dickerson R. E. How proteins recognize the TATA box. J Mol Biol. 1996 Aug 16;261(2):239–254. doi: 10.1006/jmbi.1996.0456. [DOI] [PubMed] [Google Scholar]
  26. Kerppola T. K., Curran T. Fos-Jun heterodimers and Jun homodimers bend DNA in opposite orientations: implications for transcription factor cooperativity. Cell. 1991 Jul 26;66(2):317–326. doi: 10.1016/0092-8674(91)90621-5. [DOI] [PubMed] [Google Scholar]
  27. Kim J. L., Burley S. K. 1.9 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG. Nat Struct Biol. 1994 Sep;1(9):638–653. doi: 10.1038/nsb0994-638. [DOI] [PubMed] [Google Scholar]
  28. Kim J. L., Nikolov D. B., Burley S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature. 1993 Oct 7;365(6446):520–527. doi: 10.1038/365520a0. [DOI] [PubMed] [Google Scholar]
  29. Kim Y. C., Grable J. C., Love R., Greene P. J., Rosenberg J. M. Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing. Science. 1990 Sep 14;249(4974):1307–1309. doi: 10.1126/science.2399465. [DOI] [PubMed] [Google Scholar]
  30. Kim Y., Geiger J. H., Hahn S., Sigler P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature. 1993 Oct 7;365(6446):512–520. doi: 10.1038/365512a0. [DOI] [PubMed] [Google Scholar]
  31. Klimasauskas S., Kumar S., Roberts R. J., Cheng X. HhaI methyltransferase flips its target base out of the DNA helix. Cell. 1994 Jan 28;76(2):357–369. doi: 10.1016/0092-8674(94)90342-5. [DOI] [PubMed] [Google Scholar]
  32. Klug A., Jack A., Viswamitra M. A., Kennard O., Shakked Z., Steitz T. A. A hypothesis on a specific sequence-dependent conformation of DNA and its relation to the binding of the lac-repressor protein. J Mol Biol. 1979 Jul 15;131(4):669–680. doi: 10.1016/0022-2836(79)90196-7. [DOI] [PubMed] [Google Scholar]
  33. Koo H. S., Crothers D. M. Chemical determinants of DNA bending at adenine-thymine tracts. Biochemistry. 1987 Jun 16;26(12):3745–3748. doi: 10.1021/bi00386a070. [DOI] [PubMed] [Google Scholar]
  34. Künne A. G., Meierhans D., Allemann R. K. Basic helix-loop-helix protein MyoD displays modest DNA binding specificity. FEBS Lett. 1996 Aug 5;391(1-2):79–83. doi: 10.1016/0014-5793(96)00707-7. [DOI] [PubMed] [Google Scholar]
  35. Lassar A., Münsterberg A. Wiring diagrams: regulatory circuits and the control of skeletal myogenesis. Curr Opin Cell Biol. 1994 Jun;6(3):432–442. doi: 10.1016/0955-0674(94)90037-x. [DOI] [PubMed] [Google Scholar]
  36. Ma P. C., Rould M. A., Weintraub H., Pabo C. O. Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell. 1994 May 6;77(3):451–459. doi: 10.1016/0092-8674(94)90159-7. [DOI] [PubMed] [Google Scholar]
  37. Mao Z., Nadal-Ginard B. Functional and physical interactions between mammalian achaete-scute homolog 1 and myocyte enhancer factor 2A. J Biol Chem. 1996 Jun 14;271(24):14371–14375. doi: 10.1074/jbc.271.24.14371. [DOI] [PubMed] [Google Scholar]
  38. Martin J. F., Miano J. M., Hustad C. M., Copeland N. G., Jenkins N. A., Olson E. N. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing. Mol Cell Biol. 1994 Mar;14(3):1647–1656. doi: 10.1128/mcb.14.3.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Meierhan D., el-Ariss C., Neuenschwander M., Sieber M., Stackhouse J. F., Allemann R. K. DNA binding specificity of the basic-helix-loop-helix protein MASH-1. Biochemistry. 1995 Sep 5;34(35):11026–11036. doi: 10.1021/bi00035a008. [DOI] [PubMed] [Google Scholar]
  40. Molkentin J. D., Black B. L., Martin J. F., Olson E. N. Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell. 1995 Dec 29;83(7):1125–1136. doi: 10.1016/0092-8674(95)90139-6. [DOI] [PubMed] [Google Scholar]
  41. Molkentin J. D., Firulli A. B., Black B. L., Martin J. F., Hustad C. M., Copeland N., Jenkins N., Lyons G., Olson E. N. MEF2B is a potent transactivator expressed in early myogenic lineages. Mol Cell Biol. 1996 Jul;16(7):3814–3824. doi: 10.1128/mcb.16.7.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Norman C., Runswick M., Pollock R., Treisman R. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell. 1988 Dec 23;55(6):989–1003. doi: 10.1016/0092-8674(88)90244-9. [DOI] [PubMed] [Google Scholar]
  43. Olson E. N. MyoD family: a paradigm for development? Genes Dev. 1990 Sep;4(9):1454–1461. doi: 10.1101/gad.4.9.1454. [DOI] [PubMed] [Google Scholar]
  44. Passmore S., Maine G. T., Elble R., Christ C., Tye B. K. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells. J Mol Biol. 1988 Dec 5;204(3):593–606. doi: 10.1016/0022-2836(88)90358-0. [DOI] [PubMed] [Google Scholar]
  45. Patel L., Abate C., Curran T. Altered protein conformation on DNA binding by Fos and Jun. Nature. 1990 Oct 11;347(6293):572–575. doi: 10.1038/347572a0. [DOI] [PubMed] [Google Scholar]
  46. Pellegrini L., Tan S., Richmond T. J. Structure of serum response factor core bound to DNA. Nature. 1995 Aug 10;376(6540):490–498. doi: 10.1038/376490a0. [DOI] [PubMed] [Google Scholar]
  47. Pollock R., Treisman R. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes Dev. 1991 Dec;5(12A):2327–2341. doi: 10.1101/gad.5.12a.2327. [DOI] [PubMed] [Google Scholar]
  48. Sauer R. T. Transcriptional control. Scissors and helical forks. Nature. 1990 Oct 11;347(6293):514–515. doi: 10.1038/347514b0. [DOI] [PubMed] [Google Scholar]
  49. Schultz S. C., Shields G. C., Steitz T. A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science. 1991 Aug 30;253(5023):1001–1007. doi: 10.1126/science.1653449. [DOI] [PubMed] [Google Scholar]
  50. Schwarz-Sommer Z., Huijser P., Nacken W., Saedler H., Sommer H. Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus. Science. 1990 Nov 16;250(4983):931–936. doi: 10.1126/science.250.4983.931. [DOI] [PubMed] [Google Scholar]
  51. Shatzky-Schwartz M., Arbuckle N. D., Eisenstein M., Rabinovich D., Bareket-Samish A., Haran T. E., Luisi B. F., Shakked Z. X-ray and solution studies of DNA oligomers and implications for the structural basis of A-tract-dependent curvature. J Mol Biol. 1997 Apr 4;267(3):595–623. doi: 10.1006/jmbi.1996.0878. [DOI] [PubMed] [Google Scholar]
  52. Shore P., Sharrocks A. D. The MADS-box family of transcription factors. Eur J Biochem. 1995 Apr 1;229(1):1–13. doi: 10.1111/j.1432-1033.1995.tb20430.x. [DOI] [PubMed] [Google Scholar]
  53. Sitlani A., Crothers D. M. Fos and Jun do not bend the AP-1 recognition site. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3248–3252. doi: 10.1073/pnas.93.8.3248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sommer L., Shah N., Rao M., Anderson D. J. The cellular function of MASH1 in autonomic neurogenesis. Neuron. 1995 Dec;15(6):1245–1258. doi: 10.1016/0896-6273(95)90005-5. [DOI] [PubMed] [Google Scholar]
  55. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  56. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weintraub H. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell. 1993 Dec 31;75(7):1241–1244. doi: 10.1016/0092-8674(93)90610-3. [DOI] [PubMed] [Google Scholar]
  58. Weiss M. A., Ellenberger T., Wobbe C. R., Lee J. P., Harrison S. C., Struhl K. Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature. 1990 Oct 11;347(6293):575–578. doi: 10.1038/347575a0. [DOI] [PubMed] [Google Scholar]
  59. Wing R., Drew H., Takano T., Broka C., Tanaka S., Itakura K., Dickerson R. E. Crystal structure analysis of a complete turn of B-DNA. Nature. 1980 Oct 23;287(5784):755–758. doi: 10.1038/287755a0. [DOI] [PubMed] [Google Scholar]
  60. Winkler F. K., Banner D. W., Oefner C., Tsernoglou D., Brown R. S., Heathman S. P., Bryan R. K., Martin P. D., Petratos K., Wilson K. S. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 1993 May;12(5):1781–1795. doi: 10.2210/pdb4rve/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  62. Yanofsky M. F., Ma H., Bowman J. L., Drews G. N., Feldmann K. A., Meyerowitz E. M. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature. 1990 Jul 5;346(6279):35–39. doi: 10.1038/346035a0. [DOI] [PubMed] [Google Scholar]
  63. Yee S. P., Rigby P. W. The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev. 1993 Jul;7(7A):1277–1289. doi: 10.1101/gad.7.7a.1277. [DOI] [PubMed] [Google Scholar]
  64. Yu Y. T., Breitbart R. E., Smoot L. B., Lee Y., Mahdavi V., Nadal-Ginard B. Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors. Genes Dev. 1992 Sep;6(9):1783–1798. doi: 10.1101/gad.6.9.1783. [DOI] [PubMed] [Google Scholar]
  65. Zinkel S. S., Crothers D. M. Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein. Biopolymers. 1990 Jan;29(1):29–38. doi: 10.1002/bip.360290106. [DOI] [PubMed] [Google Scholar]

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

RESOURCES