Abstract 5207: Functions of nucleolin in its interactions with the MYC promoter G-quadruplex

Abstract

Abstract DNA G-quadruplexes (G4s) are four-stranded non-canonical secondary structures formed in guanine-rich DNA sequences with functional significance and are found to be specifically enriched in the transcriptional regulatory regions of cancer-related genes. In particular, c-MYC, one of the most commonly deregulated genes in human cancers, has a DNA G4 motif in its proximal promoter which functions as a transcription inhibitor and can be targeted by G-quadruplex-interactive compounds, and is thus considered as an attractive target for cancer therapeutics. Nucleolin is overexpressed in cancer cells and is shown to be an important transcriptional regulator. Nucleolin has been found to specifically bind the c-MYC promoter G-quadruplex and to be involved in the regulation of MYC transcription, however, little is known about how the c-MYC promoter G-quadruplex interacts with and is regulated by nucleolin. By using AcGFP-tagged nucleolin, we observed nucleolin is mainly present in the cell nucleolus and nucleoplasm. We show that the nucleolin protein interacts to the MYC promoter region in vivo using chromatin immunoprecipitation assay coupled with next-generation sequencing analysis (ChIP-seq) in cancer cells. To show the association of nucleolin with G4s in vivo, we treated MCF7 cells with a G4-stabilizing ligand and monitored cellular localization of nucleolin and G4 sites using an immunofluorescent staining assay. Within 24 hr after treatment, we observed a large increase of endogenous G4 structures as well as a rapid cellular translocation of nucleolin from the cell nucleolus to the cell nucleoplasm. Importantly, the nucleolin foci were significantly colocalized with the G4 foci, suggesting the direct interaction of nucleolin and G4s in vivo. In the meantime, both MYC mRNA and protein levels were significantly reduced, indicating the nucleolin/MYC G4 interaction represses MYC transcription. To understand the interactions of nucleolin with the MYC G4 structure, we prepared various nucleolin protein constructs and studied its interactions with the MycG4 structures in vitro using NMR, EMSA, fluorescence, CD, AUC, and UV-crosslinking methods. We found that nucleolin specifically recognizes the c-MYC promoter G-quadruplex and forms a 1:1 complex with MycG4. Importantly, MycG4 maintains its G4 structure in the nucleolin-MycG4 complex. Additionally, CD melting of the MYC G4-nucleolin complex showed that MYC G4 is stabilized by nucleolin by more than 10 Celsius. In a conclusion, our in vitro and in vivo results provide important insights into the nucleolin protein interactions with the MYC promoter G4 structure and the G4-associated biological functions of nucleolin in cancer cells. Citation Format: Guanhui Wu, Luying Chen, Clement Lin, Buket Onel, Danzhou Yang. Functions of nucleolin in its interactions with the MYC promoter G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5207.