Solution Structure of a MYC Promoter G-Quadruplex with 1:6:1 Loop Length

Abstract

The important MYC oncogene is deregulated in many cancer cells and comprises one of the most prominent G-quadruplex (G4) forming sequences in its promoter regions, the NHE III₁ motif. Formation of G4s suppresses MYC transcription and can be modulated by drug binding, establishing these DNA structures as promising targets in cancer therapy. The NHE III₁ motif can fold into more than one parallel G4s, including 1:2:1 and 1:6:1 loop length conformers, with the 1:2:1 conformer shown as the major species under physiological conditions in solution. However, additional factors such as protein interactions may affect the cellular folding equilibrium. Nucleolin, a protein shown to bind MYC G4 and repress MYC transcription, is reported herein to preferably bind to the 1:6:1 loop length conformer suggesting a physiological significance of this species. The high-resolution NMR solution structure of the 1:6:1 conformer is determined, which reveals a 5'-capping structure distinctive from the 1:2:1 form, with the 6 nt central loop playing an essential role for this specific capping structure. This suggests that each parallel G-quadruplex likely adopts unique capping and loop structures determined by the specific central loop and flanking sequences. The resulting structural information at the molecular level will help to understand protein recognition of different G4s, contribution of G4 polymorphism to gene regulation, and to rationally design small molecules selectively targeting the 1:6:1 MYC G4.

Figure 1

(A) Schematic structure of the Myc1245 G4. (B) Sequence of the MycPu27 motif and its derivatives. (C) Competition electrophoretic mobility shift assay (EMSA) of Myc1245_14T and Myc2345 binding to nucleolin. [³²P-Myc1245_14T] = 10 nM, [nucleolin] = 600 nM, competing [Myc2345] or [Myc1245_14T] = 1 μM. (D) 1D NMR spectra showing the imino region of the different sequences with 100 mM K⁺, pH 7 at 25 °C unless otherwise stated.

Figure 2

(A) 2D NOESY spectral region of Myc1245 with 300 ms mixing time showing the sequential H8–H1′ contacts traced by solid lines. Missing NOE cross peaks are marked with an asterisk. 2D ¹H–¹³C HSQC of Myc1245 showing the (B) H6–C6/H8–C8 peaks for all bases and the (C) adenine H2–C2 contacts. All spectra measured at 25 °C with 100 mM K⁺, pH 7.

Figure 3

Superposition of the 10 lowest energy structures for Myc1245 (PDB: 6NEB).

Figure 4

(A) Top view of the ATG triad covering the 5′-tetrad and (B) complete 5′-capping structure. (C + D) Side views of the central loop emphasizing its two parts. (E) Top view of the base pair covering the 3′-tetrad and (F) complete 3′-capping structure.