Identifying G-quadruplex-interacting proteins in cancer-related gene promoters

Abstract

G-quadruplexes (G4s) are noncanonical DNA or RNA secondary structures involved in numerous biological processes. Their recognition by G4-related proteins (G4RPs) is essential for modulating biological pathways, particularly those associated with transcription and cancer progression. Identifying G4RPs is crucial for understanding their role in diseases like cancer, as these proteins may represent promising therapeutic targets. In this study, a proteomic-based fishing-for-partners approach was employed to identify putative interactors of G4-forming DNA sequences from the promoter regions of cancer-related genes DAP, HIF-1α, JAZF-1, and PDGF-A. A total of eighty-six G4RPs were identified, including nineteen known RNA and/or DNA G4 interactors. Notably, fourteen proteins were identified as potential interactors of all four investigated G4-forming DNA, seven of which were novel G4RPs. Direct interactions with G4s were validated for five of these proteins (AHNAK, GAPDH, HNRNP M, LMNA, and PPIA) using surface plasmon resonance experiments, which showed nanomolar binding affinities. This study not only validated known G4RPs but also led to the discovery of new G4/protein interactions, providing the basis for further investigation into their biological significance and potential implications in disease-associated pathways.

Fig. 1. DNA sequences from the DAP, HIF-1α, JAZF-1, and PDGF-A gene promoters form G4s.

Bar graphs for fluorescence enhancement of (A) ThT and (B) NMM in the presence of the tested DNA sequences. Positive controls (AS1411, c-KIT2, c-MYC, and mTEL₂₄) and negative controls (GT15 and Hrp₂₀) were included for comparison. C CD spectra of DAP, HIF-1α, JAZF-1, PDGF-A, and GT15 sequences at 20 °C, indicating the formation of parallel G4s for the gene promoter sequences. D Non-denaturing polyacrylamide gel electrophoresis of DAP, HIF-1α, JAZF-1, PDGF-A, and AS1411 sequences at 50 µM. Error bars were generated based on the standard deviation of the data and are displayed in the panels where applicable.

Fig. 2. Schematic overview of the workflow for MS-based identification of G4-interacting proteins.

Nuclear extracts were prepared from U2OS cells and incubated with the biotin-tagged unstructured control sequence (biotin-GT15). Protein-loaded control DNA was then bound to magnetic streptavidin beads and separated magnetically. The unbound protein fraction was subsequently incubated with biotinylated G4-forming sequences. Protein-loaded G4s were bound to magnetic beads and separated. Finally, G4-captured proteins, as well as GT15-interacting proteins, were subjected to sample preparation and identified by MS analysis.

Fig. 3. Profiling of the 86 proteins identified as putative G4RPs.

A Overlap between enriched DAP, HIF-1α, JAZF-1, and PDGF-A G4RPs obtained by pull-down experiments. B, C Percentage distribution of (B) biological processes and (C) molecular functions of the putative G4RPs obtained by DAVID software. Proteins enriched in Gene Ontology biological processes are highlighted in bold in (A).

Fig. 4. SPR sensorgrams for protein interaction with the G4-forming DNA sequences.

Time evolution SPR sensorgrams obtained at 25 °C by injections of various concentrations of DAP, HIF-1α, JAZF-1, and PDGF-A G4s on the chip-immobilized AHNAK protein, with a flow rate of 30 μl/min. The sensorgrams are shown as black lines and their respective fits as red lines.