G-quadruplex ligands mediate downregulation of DUX4 expression

Abstract

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV–Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

Systemic antisense therapeutics inhibiting DUX4 expression ameliorates FSHD-like pathology in an FSHD mouse model

“…Clinical management involving physiotherapies, vision and hearing aids, orthopedic interventions, pain and fatigue management or surgical scapular fixation has shown some clinical benefit and improved the quality of life for FSHD patients ( 49 , 50 ). Since aberrant expression of the DUX4 gene has been extensively reported as the main causative factor of FSHD ( 9–14 , 20 ), pre-clinical strategies silencing DUX4 expression have shown promise for FSHD treatment ( 20 , 22 , 24 , 29 , 41 , 51–53 ). Among antisense approaches, those targeting the pLAM region of DUX4 3′UTR, including ours, have provided the best down-regulatory effect ( 20 , 24 , 29 , 30 ).…”

Therapeutic Strategies Targeting DUX4 in FSHD

“…Berberine, an alkaloid that can be extracted from a variety of plants and previously shown to bind G-quadruplex DNA and RNA structures [39,40], was used to inhibit DUX4 expression. A reduction of DUX4 mRNA was observed in FSHD cells but not in vivo after a local injection of an adeno associated virus-DUX4 (AAV-DUX4) followed by repeated intraperitoneal injections of Berberine [38]. However, in vivo, a reduction of the DUX4 protein was observed.…”

“…Recently, G-quadruplex (GQ) structures, which are formed in DNA or RNA that are rich in guanine through the formation of Hoogsten hydrogen bonds between bases (for review see [37]), were identified within the enhancer, promoter, and DUX4 transcripts [38]. Berberine, an alkaloid that can be extracted from a variety of plants and previously shown to bind G-quadruplex DNA and RNA structures [39,40], was used to inhibit DUX4 expression.…”

“…DUX4 mRNA expression in skeletal muscle was validated by DUX4 quantification by RT-PCR and immunolabelling analysis. Since then, several studies aiming at reducing the DUX4-mediated toxicity have been tested following the design of this mouse model [38,42,43].…”

Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases

“…Hyaluronic acid has been reported as a signaling molecule in the mechanism of DUX4-induced molecular pathologies, which is indicative of its potential to be a therapeutic target [ 118 ]. Several G-quadruplex-forming sequences were discovered in the DUX4 locus, and berberine, a G-quadruplexes ligand, later proved to suppress DUX4 expression in patient-derived myoblasts and increase muscle strength in mouse models [ 119 ]. Although the picture is unlikely to be complete and a greater discovery of DUX4 expression factors are needed so that researchers could establish a more comprehensive pathogenic mechanism, these findings can still serve as novel therapeutic targets to treat FSHD.…”