Abstract
Abstract Deregulation of bcl-2, an anti-apoptotic protein, is associated with several human diseases such as tumorigenesis, neurodegeneration, and autoimmune deficiencies. DNA G-quadruplex secondary structures that form in the G-rich proximal promoter regions have been found to play an important role in gene transcriptional regulation. We discovered a novel GC-rich sequence in the immediate proximal P1 promoter of the bcl-2 gene (bcl2P1Pu29), which forms a thermodynamically stable, intramolecular G-quadruplex (bcl2P1G4) that represses bcl-2 gene transcription. Dimethyl sulfate footprinting studies indicated that this bcl2 P1 promoter G-rich region forms G-quadruplexes under physiologically relevant ionic conditions. This was shown by clear DMS protection of guanine N7, which is characteristic of G-quadruplex formation. NMR spectroscopic study of bcl2P1Pu29 showed the formation of a well-defined major G-quadruplex structure under physiologically relevant ionic conditions, as evident by sharp peaks in the imino proton region. Interestingly, NMR data also indicated the presence of Watson-Crick base-pairing, suggesting a potential unique loop conformation. Circular dichroism (CD) studies suggested that bcl2P1G4 adopts parallel-stranded folding. Both NMR and CD data indicated a monomeric structure of bcl2P1G4 with high thermo-stability. Using a luciferase assay, we have shown that TMPyP4, a planar cationic G-quadruplex-interactive compound, significantly inhibits the promoter activity through this G-rich bcl-2 P1 promoter region, while TMPyP2, a positional isomer of TMPyP4 which is a poor G-quadruplex-interactive compound, did not show a significant effect. Our results suggest that this novel intramolecular G-quadruplex formed in the bcl-2 proximal promoter region appears to be a suppressor of gene transcription, which could be a potential molecular target for small molecules to modulate bcl-2 gene transcription. Citation Format: Buket Onel, Megan Carver, Danzhou Yang. A novel G-quadruplex formed in the proximal P1 promoter of bcl-2 gene is a gene suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2216. doi:10.1158/1538-7445.AM2015-2216
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