G.P.109 Elucidation of DUX4 binding site affinities by SELEX-Seq

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disorder that most commonly affects specific muscles of the face, shoulder girdle, and limbs and has no effective treatment. The relative lack of focus on targeted FSHD therapies existed because the pathogenic mechanisms underlying the disease were not well understood. The field may have reached a tipping point recently with the emergence of a new FSHD pathogenesis model involving mis-expression of the pro-apoptotic DUX4 transcription factor. DUX4 belongs to the homeodomain family of proteins that bind a common AT-rich core motif. Two previous studies reported DUX4 binding sites using ChIP-seq and EMSA methodology. However, in our preliminary work using gel shifts we found that the DUX4 protein was capable of binding numerous homeodomain sites, suggesting the protein may be “sticky”. We therefore set out to better characterize DUX4 binding sequences and their relative affinities with respect to other double homeodomain proteins. To do this, we developed a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using human DUX4 protein. SELEX provides an unbiased method to determine binding motifs, and has been used to ascertain novel binding sites within genomes. Briefly, this method involved incubating purified full-length DUX4 protein with a randomized, 27-mer double-stranded oligonucleotide pool, washing unbound sequences, and PCR-amplifying eluted, DUX4-enriched binding sites. High-throughput Illumina sequencing in enriched samples was used to identify DUX4 binding motifs. We are now using bioinformatic analysis to search for these DUX4 binding sites in promoters of genes changed in FSHD patient samples and mouse muscles expressing our previously described AAV·DUX4 vectors. This study is an important early step in identifying DUX4-modulated target genes in muscle, and may pinpoint relevant pathways for targeted FSHD therapeutics, downstream of DUX4.