Abstract
Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -Å resolution crystal structure reveals that human MYC amino acids 98–111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115–124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.
Similar content being viewed by others
Data availability
Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under PDB 6E16 and PDB 6E24. Mass spectrometry data have been deposited to MassIVE under accession number: MSV000083984. Source data for Fig. 1b and Fig. 4b,d−f are available with the paper online.
References
Zeller, K. I., Jegga, A. G., Aronow, B. J., O’Donnell, K. A. & Dang, C. V. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets. Genome Biol. 4, R69 (2003).
Pijnappel W. W. M. P. et al. A central role for TFIID in the pluripotent transcription circuitry. Nature 495, 516–519 (2013).
Papai, G., Weil, P. A. & Schultz, P. New insights into the function of transcription factor TFIID from recent structural studies. Curr. Opin. Genet. Dev. 21, 219–224 (2011).
Cianfrocco M. A. et al. Human TFIID binds to core promoter DNA in a reorganized structural state. Cell 152, 120–131 (2013).
Louder, R. K. et al. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531, 604–609 (2016).
Tu, W. B et al. MYC interacts with the G9a histone methyltransferase to drive transcriptional repression and tumorigenesis. Cancer Cell 34, 579–595.e8 (2018).
Frank, S. R., Schroeder, M., Fernandez, P., Taubert, S. & Amati, B. Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes Dev. 15, 2069−2082 (2001).
Meyer, N. & Penn, L. Z. Reflecting on 25 years with MYC. Nat. Rev. Cancer 8, 976–990 (2008).
Kalkat, M. et al. MYC deregulation in primary human cancers. Genes (Basel) 8, E151 (2017).
Kato, G. J., Barrett, J., Villa-Garcia, M. & Dang, C. V. H. I. V. An amino-terminal c-myc domain required for neoplastic transformation activates transcription. Mol. Cell. Biol. 10, 5914–5920 (1990).
Burgess, S. G. et al. Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc. Natl Acad. Sci. USA 113, 13726–13731 (2016).
Helander, S. et al. Pre-anchoring of Pin1 to unphosphorylated c-Myc in a fuzzy complex regulates c-Myc activity.Structure 23, 2267–2279 (2015).
Sugase, K., Dyson, H. J. & Wright, P. E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021−1025 (2007).
Andresen, C. et al. Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding. Nucleic Acids Res. 40, 6353–6366 (2012).
Kalkat, M. et al. MYC Protein interactome profiling reveals functionally distinct regions that cooperate to drive tumorigenesis. Mol. Cell 72, 836–848.e7 (2018).
Nogales, E., Louder, R. K. & He, Y. Structural insights into the eukaryotic transcription initiation machinery. Annu. Rev. Biophys. 46, 59−83 (2017).
Hantsche, M. & Cramer, P. Conserved RNA polymerase II initiation complex structure. Curr. Opin. Struct. Biol. 47, 14–22 (2017).
Patel, A. B. et al. Structure of human TFIID and mechanism of TBP loading onto promoter DNA. Science 362, eaau8872 (2018).
Lie, W. L. et al. Structures of three distinct activator-TFIID complexes. Genes Dev. 23, 1510–1521 (2009).
Coleman, R. A. et al. p53 Dynamically directs TFIID assembly on target gene promoters. Mol. Cell Biol. 37, e00085-17(2017).
Kim, Youngchang, Geiger, James, H., Hahn, Steven & Sigler, P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature 365, 512–520 (1993).
Nikolov, D. B. et al. Crystal structure of a human TATA box-binding protein/TATA element complex. Proc. Natl Acad. Sci. USA 93, 4862–4867 (1996).
Schilbach, S. et al. Structures of transcription pre-initiation complex with TFIIH and mediator. Nature 551, 204–209 (2017).
Dergai, O. et al. Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters. Genes Dev. 32, 711–722 (2018).
Khoo, S.-K., Wu, C.-C., Lin, Y.-C., Lee, J.-C. & Chen, H.-T. Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex. Mol. Cell. Biol. 34, 551−559 (2014).
Wollmann, P. et al. Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP. Nature 475, 403–407 (2011).
Xue, Y. et al. Mot1, Ino80C, and NC2 function coordinately to regulate pervasive transcription in yeast and mammals. Mol. Cell 67, 594–607.e4 (2017).
Butryn, A., Woike, S., Shetty, S. J., Auble, D. T. & Hopfner, K.-P. Crystal structure of the full Swi2/Snf2 remodeler Mot1 in the resting state. eLife 7, e37774 (2018).
Anandapadamanaban, M. et al. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation. Nat. Struct. Mol. Biol. 20, 1008–1014 (2013).
Gouge, J. et al. Redox signaling by the RNA polymerase III TFIIB-related factor Brf2. Cell 163, 1375–1387 (2015).
Ravarani, C. N. J., Chalancon, G., Breker, M., De Groot, N. S. & Babu, M. M. Affinity and competition for TBP are molecular determinants of gene expression noise. Nat. Commun. 7, 10417 (2016).
Kawakami, E., Adachi, N., Senda, T. & Horikoshi, M. Leading role of TBP in the establishment of complexity in eukaryotic transcription initiation systems. Cell Reports 21, 3941–3956 (2017).
Wright, P. E. & Dyson, H. J. Linking folding and binding. Curr. Opin. Struct. Biol. 19, 31−38 (2009).
Ferreon, A. C. M., Ferreon, J. C., Wright, P. E. & Deniz, A. A. Modulation of allostery by protein intrinsic disorder. Nature 498, 390−394 (2013).
Hermann, S., Berndt, K. D. & Wright, A. P. How transcriptional activators bind target proteins. J. Biol. Chem. 276, 40127–40132 (2001).
Barrett, J. F., Lee, L. A. & Dang, C. V. Stimulation of Myc transactivation by the TATA binding protein in promoter-reporter assays. BMC Biochem. 6, 1–15 (2005).
Fladvad, M. et al. N and C-terminal sub-regions in the c-Myc transactivation region and their joint role in creating versatility in folding and binding. J. Mol. Biol. 346, 175–189 (2005).
Maheswaran, S., Lee, H. & Sonenshein, G. E. Intracellular association of the protein product of the c-myc oncogene with the TATA-binding protein. Mol. Cell. Biol. 14, 1147–1152 (1994).
Hateboer, G. et al. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Proc. Natl Acad. Sci. USA 90, 8489–8493 (1993).
Hann, S. R. Regulation and function of non-AUG-initiated proto-oncogenes. Biology C. 76, 880–886 (1994).
Xiao, Q. et al. Transactivation-defective c-MycS retains the ability to regulate proliferation and apoptosis. Genes Dev. 12, 3803–3808 (1998).
Chang, D. W., Claassen, G. F., Hann, S. R. & Cole, M. D. The c-Myc Transactivation domain is a direct modulator of apoptotic versus proliferative signals. Mol. Cell. Biol. 20, 4309–4319 (2000).
Wasylishen, aR. et al. New model systems provide insights into Myc-induced transformation. Oncogene 30, 3727–3734 (2011).
Dingar, D. et al. ScienceDirect BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J. Proteomics 118, 95–111 (2014).
Dingar, D. et al. BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors. J. Proteomics 118, 95–111 (2015).
Coleman, R. A., Taggart, A. K. P., Benjamin, L. R. & Pugh, B. F. Dimerization of the TATA binding protein. J. Biol. Chem. 270, 13842–13849 (1995).
Mal, T. K. et al. Structural and functional characterization on the interaction of yeast TFIID subunit TAF1 with TATA-binding protein. J. Mol. Biol. 339, 681–693 (2004).
Kotani, T. et al. A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID. Proc. Natl Acad. Sci USA 97, 7178–7183 (2000).
Johansson-Åkhe, I., Mirabello, C. & Wallner, B. InterPep2: global peptide-protein docking with structural templates. bioRxiv https://doi.org/10.1101/813238 (2019).
Alam, N et al. High-resolution global peptide-protein docking using fragments-based PIPER-FlexPepDock. PLoS Comput. Biol. 13, e1005905 (2017).
Wang, C., Bradley, P. & Baker, D. Protein-protein docking with backbone flexibility. J. Mol. Biol. 373, 503−519 (2007).
Mandell, D. J., Coutsias, E. A. & Kortemme, T. Sub-angstrom accuracy in protein loop reconstruction by robotics-inspired conformational sampling. Nat. Methods 6, 551−552 (2009).
Gupta, K et al. Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID. eLife 6, e30395 (2017).
Coleman, Ra, Taggart, A. K. P., Burma, S., Chicca, J. J. & Pugh, B. F. TFIIA regulates TBP and TFIID dimers. Mol. Cell 4, 451–457 (1999).
Kamada, K. et al. Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex. Cell 106, 71–81 (2001).
Breit, S. & Schwab, M. Suppression of MYC by high expression of NMYC in human neuroblastoma cells. J. Neurosci. Res. 24, 21–28 (1989).
Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256−268 (2003).
Kabsch, W. XDS. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 125–132 (2010).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution?. Acta Crystallogr. Sect. D Biol. Crystallogr. 69, 1204–1214 (2013).
Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. Sect. D 58, 1772–1779 (2002).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Cowtan, K. Recent developments in classical density modification. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 470–478 (2010).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 235–242 (2011).
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. Sect. D Biol. Crystallogr. 62, 1002–1011 (2006).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486−501 (2010).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Section D: Biological Crystallogr. 53, 240−255 (1997).
Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 368–380 (2012).
Adams P. D. et al. PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 213–221 (2010).
. & Yang H. et al. Automated and accurate deposition of structures solved by X-ray diffraction to the Protein Data Bank. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 1833–1839 (2044).
Gildea R. J. et al. Iotbx.cif: A comprehensive CIF toolbox. J. Appl. Crystallogr. 44, 1259–1263 (2011).
Delaglio, F et al. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Mal, T. K. et al. Resonance assignments of 30 kDa complexes of TFIID subunit TAF1 with TATA-binding protein. J. Biomol. NMR 33, 76 (2005).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol 292, 195–202 (1999).
Wasylishen, A. R. et al. MYC phosphorylation at novel regulatory regions suppresses transforming activity. Cancer Res. 73, 6504–6515 (2013).
Wasylishen, A. R. et al. MYC activity is negatively regulated by a C-terminal lysine cluster. Oncogene 33, 1066–1072 (2014).
Acknowledgements
We thank Y. Ohyama for constructing plasmids and for assistance in conducting GST-pulldown assays and M. Anandapadamanaban for insightful discussions. This work was supported by grants from the Swedish Science Council, the Carl Trygger foundation, the Swedish Child Cancer Foundation and the Swedish Cancer Foundation to M.S. and by the Canadian Institutes of Health Research (FRN# 156167) to L.Z.P. Y.W. acknowledges support from a Swedish STINT grant for institutional exchange between Linköping University and University of Toronto. This work is also supported by the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome. The results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center (SBC) at beam line 19ID of the Advanced Photon Source. SBC-CAT is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.
Author information
Authors and Affiliations
Contributions
Y.W., D.R., T.K., L.Z.P. and M.S. conceived the project. Y.W. performed crystallization experiments and together with V.M. and Z.L. performed protein purification and interactions measurements in vitro. D.R. performed the BioID-MS, proximity ligation assay and cell assays, with data interpretation by D.R., B.R. and L.Z.P. D.R., Z.L., T.K. and L.Z.P. performed and evaluated the pulldown assays and co-IP assays. S.H., A.A. and M.S. performed and evaluated NMR experiments. A.W. performed surface plasmon resonance assays. V.M. subcloned and purified proteins. I.J.-Å. and B.W. carried out structural modeling. Y.W., D.R., I.J.-Å., B.W., T.K., Y.T., L.Z.P. and M.S. wrote the manuscript. All authors discussed the results and contributed to the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Figure 1 Details of crystal structures and changes in NMR intensities in the MYC:TBP:TAF1 complex.
a, Cartoon rendering of PDB_ID 6E24, highlighting TBP (green), TAF1 (cyan), and MYC (yellow). Key elements of secondary structure of TBP are shown; helices 2 and 2’ (H2, H2’) and N- and C-lobes of TBP are labelled to designate orientation. b, Cartoon rendering of PDB_ID 6E16 in the same orientation as in a. c, 2Fo-Fc electron density map (blue) of MYC peptide (in yellow backbone) in T3M3T contoured at 1.2 s with key residues labelled. d, 2Fo-Fc electron density map (blue) of MYC peptide (in orange backbone) in T1M3T contoured at 1.2 s with key residues labelled. e, Overlay of the PDB_ID 6E24 and 6E16 structures, MYC from 6E24 in yellow and MYC from 6E16 in orange. f, Overlay of the 6E24 structure with the TBP-DNA structure (1YTB), superimposing on TBP. g, NMR intensity changes (I/I0) on MYC binding to TBP:TAF1, mapped onto the TBP:TAF1 (4B0A) structure, rendered as a cartoon. Residues of TBP and TAF1 that broaden beyond detection are coloured in green and cyan, respectively. h, same as g, but rotated 90º.
Supplementary Figure 2 Evaluation of affinity of MYC and MYC V111D to TBP-TAF1 complex using biolayer interferometry.
Biolayer interferometry binding studies performed with MYC95–158 and MYC95-158-V111D and TBP-TAF1. Three independent biological replicates are shown. GST tagged TBP-TAF1 protein was immobilized on the biosensor tip and incubated with MYC95-158 protein over a range of concentrations. 1:1 Langmuir fit was used for equilibrium KD measurement determination with the signal response time ranging from 110-115 s.
Supplementary Figure 3 Evaluation of affinity of MYC to TBP-TAF1 mutants using Surface Plasmon Resonance (SPR).
SPR binding studies performed with MYC92-167 and TBP-TAF1 mutants (Y19A, F23A, F27A, L30A). TBP-TAF1 proteins were immobilized on the biosensor tip and incubated with MYC92-167 proteins over a range of concentrations. 1:1 Langmuir fit was used for the determination of equilibrium KD.
Supplementary Figure 4 Evaluation of affinity of MYC mutants to TBP using biolayer interferometry measurements.
Biolayer interferometry binding studies performed with MYC95-158 mutants (DDDE/K, FF/A) and TBP61-240. Three independent biological replicates are shown. TBP protein was immobilized on the biosensor tip and incubated with MYC95-158 over a range of concentrations. 1:1 Langmuir fit was used for equilibrium KD measurement determination with the signal response time ranging from 110-115 s.
Supplementary Figure 5 Docking of the MYC-TBP anchor motif to TBP.
a, Sequence of docked MYC-TBP anchor motif (coloured as in Fig. 4b). b,c,d: Evaluation parameters of the top 10 predicted models: b, Rosetta reweighted energy score. c, The DDDE hydrogen bond index denotes the total number of side chain hydrogen bonds from D118, D120, D121 and E122 to TBP. d, The MYC-F(115,124) burial index denotes the degree of side chain burial into TBP of the most buried aromatic in the MYC-TBP anchor motif in the respective models (MYC-F114 or MYC-F124). e,f,g: Structural comparison of the TBP-docked MYC model (coloured as in Fig. 4e) with similar TBP anchor motifs bound to TBP: e, BRF1 (PDB: 1NGM), f, BRF2 (PDB: 5N9G), g, TAF1(TAND2) (PDB: 4B0A), coloured blue where lighter colouring indicates positioning of negatively charged residues, buried aromatics are shown as sticks (purple), and N and/or C-termini are indicated.
Supplementary Figure 6 Characterization of MYC and MYCS mutants in biological activity assays.
a, Expression of MYC mutants (c-MYC, ΔMBII, MYCS, MYCSΔMBII) tagged with V5-epitope in TET21N cells, confirmed by Immunoblot analysis with V5 antibody, and β-actin antibody as loading control. b, Soft-agar assay, conducted for MYC mutants in cells expressing either Empty Vector (EV) or V5-tagged MYC or the MYCS or MYC without MBII (ΔMBII) or MYCSΔMBII. Average colony numbers for TET21N cells expressing either EV, MYC or the MYC mutants panel (normalized to MYC counts), seeded in soft-agar conditions and treated with 1µg/mL doxycycline-containing media prior to image collection and quantification. Error bars indicate SD from biological triplicate (n=3), *p< 0.05, One-way ANOVA test, Dunnett’s multiple comparisons correction. c, Sample images for the soft-agar analysis. d, Q-RT-PCR analysis of differentially regulated nucleolin (NCL) comparing different mutants to MYC. One-way ANOVA, *p<0.05, ***p<0.001 (n=3). e, Q-RT-PCR analysis of differentially regulated lactate dehydrogenase (LDHA) comparing different mutants to MYC. One-way ANOVA, *p<0.05, ***p<0.001 (n=3).
Supplementary information
Supplementary Information
Supplementary Figures 1−6
Supplementary Data Set 1
Full-sized original western blots related to Figures 1 and 3.
Source data
Rights and permissions
About this article
Cite this article
Wei, Y., Resetca, D., Li, Z. et al. Multiple direct interactions of TBP with the MYC oncoprotein. Nat Struct Mol Biol 26, 1035–1043 (2019). https://doi.org/10.1038/s41594-019-0321-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41594-019-0321-z
- Springer Nature America, Inc.
This article is cited by
-
A druggable conformational switch in the c-MYC transactivation domain
Nature Communications (2024)
-
Cerebrospinal fluid proteomics in patients with Alzheimer’s disease reveals five molecular subtypes with distinct genetic risk profiles
Nature Aging (2024)
-
Lobetyolin inhibits cell proliferation and induces cell apoptosis by downregulating ASCT2 in gastric cancer
Cytotechnology (2023)
-
Trimeric complexes of Antp-TBP with TFIIEβ or Exd modulate transcriptional activity
Hereditas (2022)
-
Cryo-EM structure of human SAGA transcriptional coactivator complex
Cell Discovery (2022)