An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution

Abstract

We report the first G-quadruplex structure formed in the promoter region of the human bcl-2. Bcl-2 is a potent oncoprotein that functions as an inhibitor of cell apoptosis and has been found to be aberrantly overexpressed in a wide range of human tumors. A highly GC-rich region upstream of the P1 promoter plays an important role in the regulation of the transcriptional activity of the bcl-2 oncogene. The purine-rich strand of this region contains multiple runs of guanines and can form three distinct intramolecular G-quadruplexes in K+-containing solution. Of these, the G-quadruplex formed within the middle four consecutive guanine runs has been shown to be the most stable G-quadruplex structure, while it is also a mixture of loop isomers. The predominant G-quadruplex structure formed in this region was studied by NMR. Our results demonstrate a novel folding of a unique intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands. This G-quadruplex structure contains three G-tetrads connected with a single-nucleotide double-chain-reversal side loop and two lateral loops. The first three-nucleotide CGC loop in the bcl-2 promoter sequence forms a lateral loop, as opposed to a double-chain-reversal side loop observed in a similar sequence in the c-MYC promoter, which appears to largely determine the overall folding of the bcl-2 G-quadruplex. Furthermore, both the bcl-2 and c-MYC promoter sequences contain the G3NG3 sequence motif, which forms a stable double-chain-reversal, parallel-stranded structural motif. This predominant bcl-2 G-quadruplex represents an attractive novel target for the design of new anticancer drugs that specifically modulate bcl-2 gene expression.

Figure 1

(A) The promoter sequence of the bcl-2 gene and its modifications. The top sequence is the wild-type bcl-2 39-mer sequence. The six G-runs are underlined and numbered using Roman numerals. Bcl2MidG4Pu23 represents the 23mer sequence containing the middle four consecutive runs of guanines, which forms the most stable G-quadruplex structure. Bcl2MidG4Pu23-G15T/G16T represents the mutant 23mer with 15,16-G-to-T mutations. (B) Imino and aromatic regions of 1D ¹H NMR spectra of bcl2MidG4Pu23 (upper) and bcl2MidG4Pu23-G15T/G16T (lower), at 7 °C, 20 mM K-phosphate, 40 mM KCl, pH 7.0.

Figure 2

CD spectra of bcl2MidG4Pu23 (WT) and bcl2MidG4Pu23-G15T/G16T (M1) in the absence and presence of 100 mM KCl.

Figure 3

(A) Imino proton assignments of bcl2MidG4Pu23-G15T/G16T using 1D ¹⁵N-filtered experiments on site-specific labeled oligonucleotides. The imino proton of G1 is assigned using elimination method, in combination with the exchangeable-proton 2D-NOESY (see Figure 3C). (B) (Top) A G-tetrad with H1-H1 and H1–H8 connectivity pattern detectable in NOESY experiments. (Bottom) Schematic drawing of the folding topology of the bcl2MidG4Pu23-G15T/G16T G-quadruplex. Red boxes represent guanines with anti configuration, and blue boxes represent guanines with syn configuration. (C) H1–H8 region (top) and H1-H1 region (bottom) of 2D-NOESY spectrum of bcl2MidG4Pu23-G15T/G16T in H₂O at 25 °C. Red boxes represent intra-tetrad connectivity, green boxes represent inter-tetrad connectivity, and blue boxes represent sequential connectivity.