Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells

Abstract

Guanine-rich DNA of a particular sequence adopts four-stranded structural forms known as G-quadruplex or G4 DNA. Though in vitro formation of G4 DNA is known for several years, in vivo presence of G4 DNA was only recently noted in eukaryote telomeres. Recent bioinformatics analyses showing prevalence of G4 DNA within promoters of human and related species seems to implicate G4 DNA in a genome-wide cis-regulatory role. Herein we demonstrate that G4 DNA may present regulatory sites on a genome-wide scale by showing widespread effect on gene expression in response to the established intracellular G4 DNA-binding ligands. This is particularly relevant to genes that harbor conserved potential G4 DNA (PG4 DNA) forming sequence across human, mouse and rat promoters of orthologous genes. Genes with conserved PG4 DNA in promoters show co-regulated expression in 79 human and 61 mouse normal tissues (z-score > 3.5; P < 0.0001). Conservation of G4 DNA across related species also emphasizes the biological importance of G4 DNA and its role in transcriptional regulation of genes; shedding light on a relatively novel mechanism of regulation of gene expression in eukaryotes.

Figure 1.

Genome-wide expression analysis shows wide-spread PG4 DNA-mediated gene regulation. (A) SAM plot showing overexpressed or repressed genes at ≤20% FDR; (B) distribution of significantly differentially expressed genes (≤20% FDR) in microarray based on presence/absence of PG4 within ±1 kb from transcription start sites. (C) Validation of microarray results by quantitative RT-PCR. Ten out of twelve reactions tested gave consistent change in expression on TMPyP4 treatment, in both cell lines. All selected genes (12) were at ≤20% FDR. Transcript level could not be detected for DHCR24 gene in A549 cells.

Figure 2.

Gene expression change in presence of G4 DNA-binding ligands was consistent with TMPyP4 but not TMPyP2, which does not bind to G4 DNA. (A) Scheme showing the structure of ligands used. Quantitative RT-PCR results 48 h after treatment of A549 cells with BMVC/TpPy/TMPyP4 (B) or TMPyP4/TMPyP2 (C). Average of at least three measurements is given for each PCR.

Figure 3.

Tissue-specific expression of genes with conserved PG4 DNA in promoters. 71 genes show enriched expression (either up- or downregulated) in 72/79 human and 56/61 mouse (normal) tissues. Statistical tissue-specific expression enrichment (z-score > 3.5, P < 0.0001) is represented in pseudo-color for both human and mouse tissues. Genes (200) without PG4 DNA in ±1 kb or ±5 kb (centered at TSS) do not show enrichment of expression in 79 human tissues (Figure 1).