Examining Cooperative Binding of Sox2 on DC5 Regulatory Element Upon Complex Formation with Pax6 Through Excess Electron Transfer Assay

  • Abhijit Saha
Part of the Springer Theses book series (Springer Theses)


Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein–DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analyzing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, BrU-labeled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.


Excess electron transfer Transcription factor Tryptophan BrU-substituted DNA Cooperative effect 


  1. 1.
    Levine M, Tjian R (2003) Transcription regulation and animal diversity. Nature 424:147–151. CrossRefGoogle Scholar
  2. 2.
    Vaquerizas JM, Kummerfeld SK, Teichmann SA, Luscombe NM (2009) A census of human transcription factors: function, expression and evolution. Nat Rev Genet 10:252–263. CrossRefGoogle Scholar
  3. 3.
    Petti AA, McIsaac RS, Ho-Shing O, Bussemaker HJ, Botstein D (2012) Combinatorial control of diverse metabolic and physiological functions by transcriptional regulators of the yeast sulfur assimilation pathway. Mol Biol Cell 23:3008–3024. CrossRefGoogle Scholar
  4. 4.
    Cunha PMF, Sandmann T, Gustafson EH, Ciglar L, Eichenlaub MP, Furlong EEM (2010) Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity. PLoS Genet 6:e1001014. CrossRefGoogle Scholar
  5. 5.
    Reményi A, Lins K, Nissen LJ, Reinbold R, Schöler HR, Wilmanns M (2003) Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev 17:2048–2059. CrossRefGoogle Scholar
  6. 6.
    Ng CK, Li NX, Chee S, Prabhakar S, Kolatkar PR, Jauch R (2012) Deciphering the Sox-Oct partner code by quantitative cooperativity measurements. Nucleic Acids Res 40:4933. CrossRefGoogle Scholar
  7. 7.
    Kamachi Y, Uchikawa M, Tanouchi A, Sekido R, Kondoh H (2001) Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev 15:1272–1286. CrossRefGoogle Scholar
  8. 8.
    Narasimhan K, Pillay S, Huang YH, Jayabal S, Udayasuryan B, Veerapandian V, Kolatkar P, Cojocaru V, Pervushin K, Jauch R (2015) DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors. Nucleic Acids Res 43:1513. CrossRefGoogle Scholar
  9. 9.
    Morimura H, Tanaka S, Ishitobi H, Mikami T, Kamachi Y, Kondoh H, Inouye Y (2013) Nano-analysis of DNA conformation changes induced by transcription factor complex binding using plasmonic nanodimers. ACS Nano 7:10733. CrossRefGoogle Scholar
  10. 10.
    Cvekl A, Ashery-Padan R (2014) The cellular and molecular mechanisms of vertebrate lens development. Development 141:4432. CrossRefGoogle Scholar
  11. 11.
    Danno H, Michiue T, Hitachi K, Yukita A, Ishiura S, Asashima M (2008) Molecular links among the causative genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression. Proc Natl Acad Sci 105:5408–5413. CrossRefGoogle Scholar
  12. 12.
    Shimozaki K, Zhang CL, Suh H, Denli AM, Evans RM, Gage FH (2012) SRY-box-containing gene 2 regulation of nuclear receptor tailless (Tlx) transcription in adult neural stem cells. J Biol Chem 287:5969–5978. CrossRefGoogle Scholar
  13. 13.
    Lodato MA, Ng CW, Wamstad JA, Cheng AW, Thai KK, Fraenkel E, Jaenisch R, Boyer LA (2013) SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state. PLoS Genet 9:e1003288. CrossRefGoogle Scholar
  14. 14.
    Yamamoto S, De D, Hidaka K, Kim KK, Endo M, Sugiyama H (2014) Single molecule visualization and characterization of Sox2-Pax6 complex formation on a regulatory DNA element using a DNA origami frame. Nano Lett 14:2286–2292. CrossRefGoogle Scholar
  15. 15.
    Sontz PA, Muren NB, Barton JK (2012) DNA charge transport for sensing and signalling. Acc Chem Res 45:1792–1800. CrossRefGoogle Scholar
  16. 16.
    Andrew WA, Michael A, Stephen LM, Robert JC, Harry BG (1988) Distance dependence of photoinduced long-range electron transfer in zinc/ruthenium-modified myoglobins. J Am Chem Soc 110:435–439. CrossRefGoogle Scholar
  17. 17.
    DeRosa MC, Sancar A, Barton JK (2005) Electrically monitoring DNA repair by photolyase. Proc Natl Acad Sci USA 102:10788–10792. CrossRefGoogle Scholar
  18. 18.
    Boon EM, Livingston AL, Chimiel NH, David SS, Barton JK (2003) DNA-mediated charge transport for DNA repair. Proc Natl Acad Sci USA100, 12543–12547.
  19. 19.
    Yavin E, Boal AK, Stemp ED, Boon EM, Livingston AL, O’Shea VL, David SS, Barton JK (2005) Protein-DNA charge transport: redox activation of a DNA repair protein by guanine radical. Proc Natl Acad Sci USA 102:3546–3551. CrossRefGoogle Scholar
  20. 20.
    Kim S, Li Y, Sancar A (1992) The third chromophore of DNA photolyase: Trp-277 of Escherichia coli DNA photolyase repairs thymine dimers by direct electron transfer. Proc Natl Acad Sci USA 89:900–904CrossRefGoogle Scholar
  21. 21.
    Sancar A (2003) Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem Rev 103:2203–2237. CrossRefGoogle Scholar
  22. 22.
    BehmoarasT Toulme JJ, Hélène C (1981) A tryptophan-containing peptide recognizes and cleaves DNA at apurinic sites. Nature 292:858–859. CrossRefGoogle Scholar
  23. 23.
    Wagenknecht HA, Stemp EDA, Barton JK (2000) Evidence of electron transfer from peptides to DNA: oxidation of DNA-bound tryptophan using the flash-quench technique. J Am Chem Soc 122:1. CrossRefGoogle Scholar
  24. 24.
    Wagenknecht HA, Rajski SR, Pascaly M, Stemp ED, Barton JK (2001) Direct observation of radical intermediates in protein-dependent DNA charge transport. J Am Chem Soc 123:4400. CrossRefGoogle Scholar
  25. 25.
    Mayer-Enthart E, Kaden P, Wagenknecht HA (2005) Electron transfer chemistry between DNA and DNA-binding tripeptides. Biochemistry 44:1749–1757. CrossRefGoogle Scholar
  26. 26.
    Tashiro R, Wang AH, Sugiyama H (2006) Photoreactivation of DNA by an archaeal nucleoprotein Sso7d. Proc Natl Acad Sci USA 103:16655–16659. CrossRefGoogle Scholar
  27. 27.
    Sugiyama H, Tsutsumi Y, Saito I (1990) Highly sequence-selective photoreaction of 5-bromouracil-containing deoxyhexanucleotides. J Am Chem Soc 112:6720–6721. CrossRefGoogle Scholar
  28. 28.
    Sugiyama H, Fujimoto K, Saito I (1996) Evidence for intrastrand C2’ hydrogen abstraction in photoirradiation of 5-halouracil-containing oligonucleotides by using stereospecifically C2’-deuterated deoxyadenosine. Tetrahedron Lett 37:1805–1808. CrossRefGoogle Scholar
  29. 29.
    Sugiyama H, Tsutsumi Y, Fujimoto K, Saito I (1993) Photoinduced deoxyribose C2’ oxidation in DNA. Alkali-dependent cleavage of erythrose-containing sites via a retroaldol reaction. J Am Chem Soc 115:4443–4448. CrossRefGoogle Scholar
  30. 30.
    Saha A, Hashiya F, Kizaki S, Asamitsu S, Hashiya K, Bando T, Sugiyama H (2015) A novel detection technique of polyamide binding sites by photo-induced electron transfer in BrU substituted DNA. Chem Commun 51:14485. CrossRefGoogle Scholar
  31. 31.
    Hashiya F, Saha A, Kizaki S, Li Y Sugiyama H (2014) Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA. Nucleic Acids Res 42:13469–13473.
  32. 32.
    Lewis FD, Wu Y (2001) Dynamics of superexchange photoinduced electron transfer in duplex DNA. J Photochem Photobiol C Photochem Rev 2:1–16. CrossRefGoogle Scholar
  33. 33.
    Watanabe T, Bando T, Xu Y, Tashiro R Sugiyama H (2005) Efficient generation of 2’-deoxyuridin-5-yl at 5’-(G/C)AA(X)U(X)U-3’ (X = Br, I) sequences in duplex DNA under UV irradiation. J Am Chem Soc 127:44–45.
  34. 34.
    Watanabe T, Tashiro R, Sugiyama H (2007) Photoreaction at 5’-(G/C)AA(Br)UT-3’ sequence in duplex DNA: efficient generation of uracil-5-yl radical by charge transfer. J Am Chem Soc 129:8163–8168. CrossRefGoogle Scholar
  35. 35.
    Jolma A, Yin Y, Nitta KR, Dave K, Popov A, Taipale M, Enge M, Kivioja T, Morgunova E, Taipale J (2015) DNA-dependent formation of transcription factor pairs alters their binding specificity. Nature 527:384. CrossRefGoogle Scholar
  36. 36.
    Xu Y, Tashiro R, Sugiyama H (2007) Photochemical determination of different DNA structures. Nat Protoc 2:78–87. CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Institute CurieOrsayFrance

Personalised recommendations