Abstract Nucleolin is an important regulator of cell proliferation and has been reported to play a role in c-Myc transcriptional regulation. Elevated expression levels of nucleolin have been shown in a number of malignant cells, but how nucleolin contributes to cancer development and progression remains unknown. Recently, G-quadruplexes (G4s) have been shown in vivo to form in G-rich sequences with biological importance. Nucleolin has been reported to bind to G-quadruplexes for its biological functions. In particular, G4s have been shown to form in the MYC promoter and inhibit MYC transcription; nucleolin was found to display a remarkable binding affinity to the c-Myc promoter G4 over its best-known RNA target. CX-3543, the first-in-class G-quadruplex-targeting small molecule, was shown to induce nucleolin redistribution from the cell nucleolus to the cell nucleoplasm and apoptosis in cancer cells, which was suggested to act through the c-Myc promoter G-quadruplex. Therefore, unbiased analysis of the genomic binding sites of nucleolin in human chromatin is pivotal in understanding nucleolin's biological functions. Using chromatin immunoprecipitation (ChIP) assay, we demonstrated that nucleolin binds to the c-Myc NHE III1 region in vivo. Using an anti-nucleolin antibody, the isolated DNA from the immunoprecipitated DNA-protein complexes from HeLa cells yielded numerous c-Myc promoter sequences that were determined by real-time PCR, suggesting the direct interaction between nucleolin and the c-Myc promoter G4-forming region in vivo. G4-interactive ligands have been shown to facilitate the formation of G4 structures in cells. To elucidate the association of nucleolin with G-quadruplexes in vivo, we monitored cellular localization of nucleolin before and after G4 ligand treatments and observed a rapid cellular translocation of nucleolin from the cell nucleolus to the cell nucleoplasm. Using ChIP coupled with next-generation sequencing analysis (ChIP-seq), we identified several binding sites of nucleolin in human chromatin. The determined chromatin binding sites of nucleolin will provide insights into the biological functions of nucleolin and its relationship with G-quadruplexes in cancer cells. Citation Format: Guanhui Wu, Buket Onel, Danzhou Yang. Identifying novel chromatin binding sites of nucleolin protein in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1474.