The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor ? Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression.

ABSTRACT: The platelet-derived growth factor receptor ? (PDGFR-?) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-? promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-? NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-? pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-? is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-? NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-? NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-? promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-? i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-? promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-?-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.

SUBMITTER: Brown RV 

PROVIDER: S-EPMC5977998 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature