In-depth analysis of the mode of action of resveratrol: genome-wide characterization of G-quadruplex binding properties

Abstract

Background: Resveratrol (RSV) is one of the most studied and used biomolecules, for which many pharmacological effects targeting multiple tissues have been described. However, a common underlying mechanism driving its full pharmacological activity has not been described in detail. G-quadruplexes (G4s) are non-canonical nucleic acid structures found in regulatory genomic locations and involved in controlling gene transcription, telomere maintenance, or genome stability, among others. This study provides a genome-wide characterization of RSV G4-binding properties, explaining its multi-target traits.

Methods: Immunofluorescence assays using a nucleolar and a G4-specific antibody were used to characterize RSV cellular effects on the nucleolus and G4 stabilization. DNA damage and cell cycle analyses were performed via western blot and flow cytometry. Breaks lLbeling In Situ and Sequencing (BLISS) was used to map double strand breaks (DSB) in response to treatment, and identify G4s targeted by RSV. mRNA sequencing was used to identify changes at the transcriptional level upon treatment and relate them to a direct targeting of G4s. Biophysical assays (circular dichroism, ultraviolet-visible [UV-Vis] titration, differential scanning calorimetry, and nuclear magnetic resonance) were used to characterize RSV-G4 interactions. Lastly, luciferase-based transcription assays were performed to confirm RSV-G4 interaction in vitro and its direct influence on gene expression.

Results: In a cellular context, RSV treatment showed classic G4-ligand effects, such as nucleolar disassembly, inhibition of RNA polymerase I, DNA damage, and cell cycle arrest. RSV was shown to stabilize cellular G4s, which accumulated around double strand breaks in the promoters of differentially expressed genes. Upon treatment, G4 stabilization triggered DNA damage and controlled gene expression. The interaction between RSV and target G4s was confirmed in vitro by biophysical assays and through luciferase-based transcription assays.

Conclusions: A G4-dependent mode of action was demonstrated as the main mechanism underlying RSV pleiotropic effects, along with the identification of target genes and G4s. This in-depth analysis of the mode of action of RSV will be helpful to improve its therapeutic potential in a wide variety of health scenarios.

Keywords: DNA binding; G-quadruplex; Resveratrol; Secondary DNA structures; Small molecule.

Fig. 1

Effect of resveratrol on the nucleolus of the A375 cell line and rDNA–G4 targeting properties. A Nucleolin (NCL) staining in the control and treated group (RSV IC50 6 h). Images shown are representative of three independent experiments (n = 3). Counterstaining was performed with DAPI. Scale bar = 10 µm. B A schematic view of the rDNA organization in genomic DNA is represented in the top part. Primers targeting 5′ ETS, ITS1, ITS2, and 3′ ETS to analyze rDNA transcription are shown with red arrows. A bar diagram representing rRNA expression levels (fold change) in the control and treated group (RSV IC50 6 h) is shown in the bottom part. C FID assay bar diagram showing decreasing values of TOPRO3 + DNA fluorescence in response to RSV in four G4-containing primers from the 5′ ETS region. Error bars indicate standard deviations for three independent experiments (n = 3)

Fig. 2

Genome-wide G4 targeting and derived effects. A Nuclear G4 staining in control and treated conditions in the A375 cell line (RSV IC50 6 h). Images are representative of three independent experiments (n = 3). B Quantification of 100 nuclei per condition was performed using ImageJ in the three replicates. C ɣH2AX protein levels indicating DNA damage in the control and treated groups in the A375 cell line (RSV IC50 6 h). Images are representative of three independent experiments (n = 3). D Bar plot showing quantified and normalized protein levels relative to actin (ACT) in the three replicates. E Bar diagram for cell cycle distribution in the control and treated groups in the A375 cell line (n = 3) (RSV IC50 24 h). F Representative histograms of experiments in E. Error bars indicate standard deviations

Fig. 3

Changes in gene expression in response to RSV treatment are related to G4 targeting. A Volcano plot showing differentially expressed genes (DEGs; FDR < 0.05) with log₂FC < −1 in blue and log₂FC > 1 in red (n = 2). qPCR validated genes have been highlighted with blue and red labels. B Scatter plot showing the number of double strand breaks (DSBs) per distance range (kb) in relation to the nearest G4 [25], denoted by 0. Black dots indicate the median for each experimental group (n = 2). C Bar plot for the number of DSBs per UMI slot in response to RSV treatment and expressed as fold change. D Bar plot for the number of DSBs in each genomic region in response to RSV treatment and expressed as fold changes. E Scatter plot showing the relationship between the distance from each DSB to its nearest gene and its log₂FC (n = 2). All DEGs were considered. (F) Heatmap showing the number of described G4s [25] in the forward (+) and reverse (−) strand per distance range (kb) in relation to the nearest DSB (denoted by 0) in treated samples. All DEGs and genes with no differential expression were considered. Error bars indicate standard deviations

Fig. 4

Binding assays and luciferase-based transcription assays confirm that RSV G4-targeting affects gene expression. A CD spectra of PITPNB G4 in the presence (green line) and absence (gray line) of RSV. Arrows indicate the direction of the movement of CD peaks upon addition of RSV. B UV–Vis spectra resulting from the titration of PITPNB G4 with RSV. Arrows indicate the direction in which the absorption peak moves after each addition of RSV. C Thermal melting profiles showing excess heat capacity as a function of temperature for PITPNB with (green line) and without RSV (gray line); the arrow indicates the ligand-dependent conformational state. D Exchangeable proton region of the NMR spectra of PITPNB with RSV at different DNA:RSV ratios. E Bar plot showing the inhibition of the promoter activity of the PITPNB gene as a consequence of RSV treatment in the A375 cell line. F Bar plot showing changes in the activity of the mutated promoter of the PITPNB gene as a consequence of RSV treatment in the A375 cell line. Error bars indicate the standard deviation of three independent experiments (n = 3)