The major G-quadruplex formed in the human BCL-2 proximal promoter adopts a parallel structure with a 13-nt loop in K+ solution

Abstract

The human BCL-2 gene contains a 39-bp GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of BCL-2 gene expression. Inhibition of BCL-2 expression can decrease cellular proliferation and enhance the efficacy of chemotherapy. Here we report the major G-quadruplex formed in the Pu39 G-rich strand in this BCL-2 promoter region. The 1245G4 quadruplex adopts a parallel structure with one 13-nt and two 1-nt chain-reversal loops. The 1245G4 quadruplex involves four nonsuccessive G-runs, I, II, IV, V, unlike the previously reported bcl2 MidG4 quadruplex formed on the central four G-runs. The parallel 1245G4 quadruplex with the 13-nt loop, unexpectedly, appears to be more stable than the mixed parallel/antiparallel MidG4. Parallel-stranded structures with two 1-nt loops and one variable-length middle loop are found to be prevalent in the promoter G-quadruplexes; the variable middle loop is suggested to determine the specific overall structure and potential ligand recognition site. A limit of 7 nt in loop length is used in all quadruplex-predicting software. Thus, the formation and high stability of the 1245G4 quadruplex with a 13-nt loop is significant. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter is intriguing, suggesting a novel mechanism for gene transcriptional regulation and ligand modulation.

Figure 1

(A) The promoter sequence of the BCL-2 gene and its modifications. The top sequence is the 39-mer wild-type G-rich sequence (Pu39). The six G-runs with three or more guanines are underlined and numbered. Pu30 is the wild-type 30-mer G-rich sequence containing the I–V G-runs; the numbering used in this study is shown for Pu30. The guanine residues that are involved in the tetrad formation of the major BCL-2 G-quadruplex 1245G4 are shown in red. The mutations are shown in cyan. (B) DMS footprinting of the wild-type Pu39 with densitometric scans (left) and Pu30 (right). (C) Imino regions of 1D ¹H NMR spectra of BCL-2 promoter sequences at 25 °C in 45 mM K⁺, pH 7.0. (D) CD spectra of Pu30 sequences in 95 mM K⁺.

Figure 2

(A) A G-tetrad with detectable H1–H1 and H1–H8 NOE connectivity. (B) Imino H1 and aromatic H8 proton assignments of Pu30_3T4AA by 1D ¹⁵N-filtered experiments using site-specific labeled oligonucleotides at 25 °C. (C) Schematic drawing of the folding topology of the major G-quadruplex 1245G4 formed in the BCL-2 promoter sequence Pu30 (G = red, A = green, C = yellow). (D) H1–H1 region and (E) H1–H8 region of the 2D-NOESY spectrum of Pu30_3T4AA in H₂O at 5 °C with a mixing time of 200 ms. The proton assignments are shown on the sides. Intratetrad NOEs are in red, intertetrad NOEs in blue, sequential intertetrad NOEs in black, and NOEs with flanking bases in green. Conditions: 25 mM K-phosphate, 70 mM KCl, pH 7.0.

Figure 3

G-quadruplex-forming promoter sequences.