Abstract 2130: The novel G-quadruplex with hairpin loop formed immediately upstream of the human BCL-2 P1 promoter represses BCL-2 transcription

Abstract

Abstract The abnormal overexpression of the BCL-2 protein is linked to a large number of cancers. Elevated levels of BCL-2 are found to promote resistance to chemotherapy and radiation. Therefore, BCL-2 is considered to be an attractive target for cancer therapeutics. We have previously identified a 39-base-pair GC-rich sequence located in the major P1 promoter region of the human BCL-2 gene, whose G-rich strand (Pu39) can form two interchangeable G-quadruplex (G4) structures, a hybrid-type G-quadruplex and a parallel G-quadruplex with a 13-nt middle loop. However, in our functional study of the BCL-2 P1 promoter activity using a luciferase reporter system containing this region in tumor cells, we found that the construct with a complete G-quadruplex-knock-out Pu39 mutant is still affected by G4-interactive compounds. In this study we report a new 29-mer G-quadruplex-forming sequence, P1G4, just downstream of Pu39 and immediately upstream of the human BCL-2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. Using NMR spectroscopy, the P1G4 G-quadruplex is shown to be a novel dynamic equilibrium of two parallel structures, one regular G4 with two 1-nt loops and a 12-nt middle loop and another broken-strand G4 with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) structure in the long loop. Using dimethyl sulfate (DMS) footprinting assays, we show that G-quadruplexes can readily form in the P1G4 sequence under physiological salt conditions, and that P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions. To further understand the respective function of Pu39 and P1G4, we created luciferase-constructs driven by the BCL-2 promoter sequence including both Pu39 and P1G4, and examined their functions by knocking out either or both of them. We found P1G4 independently and predominantly represses BCL-2 transcription. Unlike the 1245 parallel G-quadruplex formed in Pu39, P1G4 structure is easily compromised by modifications in the hairpin loop, indicating the loop is crucial for the maintenance of this dynamic structure. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures, and may provide a unique target for small molecules to modulate BCL-2 gene transcription. The presence of P1G4 and Pu39 G-quadruplexes in adjacent regions suggests a mechanism for precise regulation of BCL-2 gene transcription. Citation Format: Buket Onel, Guanhui Wu, Megan Carver, Daria Timonina, Marti Larriva, Danzhou Yang. The novel G-quadruplex with hairpin loop formed immediately upstream of the human BCL-2 P1 promoter represses BCL-2 transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2130.