LAUSR

DDX5 (DEAD-box protein 5) plays an important role in cell proliferation, differentiation, and tumorigenesis. DDX5 is a founding member of the DEAD-box RNA helicase family and known as a non-processive RNA helicase. It acts as a transcriptional co-activator of many cancer-associated genes, such as MYC, however, the underlying molecular mechanism is unknown. MYC is one of the most critical oncogenes and has a DNA G-quadruplex in its proximal promoter region (MycG4) that functions as transcriptional silencer. Guanine-rich DNA and RNA sequences can form G-quadruplex, a non-canonical secondary structure. However, MycG4 is highly stable and its regulatory role in transcription requires active unfolding. Here, we report that DDX5 unfolds MycG4 with extreme efficiency, and thereby transactivates MYC expression. To understand the effects of DDX5 on MycG4, we characterized the unfolding of MycG4-DNA within the DDX5-MycG4 complex using DNA footprinting, FRET and CD spectroscopy. While DDX5 is known as a dsRNA helicase, our results showed that DDX5 is a highly active DNA G4-resolvase. Strikingly, MycG4 unfolding requires neither ATP hydrolysis nor extended loading of single-stranded flanking. Our protein-binding-ELISA experiments revealed specific and high-affinity binding of DDX5 to G4 structures regardless of whether the substrate is DNA or RNA. To elucidate the cellular functions of DDX5, we analyzed publicly available DDX5 ChIP-seq data and identified G-rich sequences in cancer cells as chromatin binding sites of DDX5. Moreover, our ChIP and Western Blot results showed that DDX5 is enriched at the MYC promoter and activates MYC transcription. Importantly, G-quadruplex interactive small molecules can inhibit the DDX5 interaction with the promoter MycG4 and DDX5-mediated transcriptional activation of the MYC gene. In addition, knock-down of DDX5 expression with DDX5-specific siRNA in cancer cells resulted in downregulation of MYC expression and sensitization to G4-interactive small molecules. In summary, we identified resolving DNA and RNA G-quadruplexes as novel function of DDX5. Our results establish that the MYC-transactivation by DDX5 is mediated through unfolding of the MYC promoter quadruplex and thereby establish a new molecular target to suppress MYC for cancer intervention. Citation Format: Guanhui Wu, Zheng Xing, Luying Chen, Elizabeth J. Tran, Danzhou Yang. DDX5 helicase resolves G-quadruplex and transactivates MYC expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3948.