G4-DNA formation and chromatin remodelling are interdependent in human cells

Abstract

DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.

Fig. 1. Small molecule G4-stabilising ligands. Molecular structures of (a) GQC-05 and (b) GTC365. Cell viability curves determined via MTS for MCF-7 cells after 72 h treatment with (c) GQC-05 and (d) GTC365.

Fig. 2. Effects of GQC-05 and GTC365 on nuclear G4 stabilisation and induction of DNA damage. (a) Representative images of immunocytochemistry of ligand-treated MCF-7 cells using BG4 (red) and γH2AX (green) antibodies, with nuclei counterstained with Hoechst 34580 (blue); Plan Apo 60×/1.40 objective; scale bar 10 μm. (b) The number of BG4 foci were counted and found to be greatest in GQC-05-treated cells, with GTC365 also increasing foci counts relative to the control (n > 90 for each sample). (c) GQC-05 also causes an increase in γH2AX foci, corresponding to instances of DNA damage, while GTC365 did not induce a significant change (n > 90 for each sample). **p < 0.01, ****p < 0.0001.

Fig. 3. ATAC-seq analysis for GQC-05- and GTC365-treated MCF-7 cells. Mean-difference (MD) plots for (a) GQC-05 and (b) GTC365 treatments with differentially accessible regions shown in red (increased accessibility) and blue (decreased accessibility). FC, fold change; CPM, counts per million. (c) Feature distribution of ATAC-seq peaks in genomic regions shows increased promoter accessibility following GQC-05 treatment. (d) Predicted G4 (pG4) counts and change in chromatin accessibility (log₂ FC) for the subset of 22 DARs located within gene promoter regions in GTC365-treated MCF-7 cells. Dots represent pG4 counts, and bars show the relative accessibility of the ATAC peak. All 21 regions with reduced accessibility contain pG4 sequences, while the single region with increased accessibility has no pG4s. (e) Distribution of ATAC peaks and pG4s in the promoter region of HIVEP1. The genomic positions of pG4 structures (green), the HIVEP1 gene (black), and ATAC peaks from the control (pink) and GTC365 (blue) ATAC-seq datasets are shown. Filtered DNA- and RNA-related (f) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and (g) Gene Ontology analysis of genes containing ATAC peaks in the promoter region, with colour corresponding to the adjusted p-value and dot size corresponding to the ratio of genes from the dataset associated with each pathway.

Fig. 4. RNA-seq analysis for GQC-05- and GTC365-treated MCF-7 cells. MD plots for (a) GQC-05 and (b) GTC365 treatments with differentially expressed genes in red (upregulated) and blue (downregulated). (c) Gene set enrichment analysis (GSEA) of the GTC365 RNA-seq data, filtered to show pathways associated with DNA associated processes. The complete data is provided in Table S3 and Fig. S5. The distribution of genes associated with each pathway is indicated (gene ranks), as is the normalised enrichment score (NES) and adjusted p-value.