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.
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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.
The authors declare no competing interests.
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.
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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.
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.
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).
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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) doi:10.1038/s41594-019-0321-z