We present the first large-scale identification of lariats—the transient branched introns that are released as a byproduct of pre-mRNA splicing. The locations of the branchpoints in these introns provide insight into the early steps of splicing. From this data set, we have developed a comprehensive model of 3′ splice-site selection, identified new mechanisms of alternative splicing and mapped the distribution of splicing factors around branchpoints.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

Facioscapulohumeral Muscular Dystrophy is a common form of muscular dystrophy that presents clinically with progressive weakness of the facial, scapular, and humeral muscles, with later involvement of the trunk and lower extremities. While typically inherited as autosomal dominant, facioscapulohumeral muscular dystrophy (FSHD) has a complex genetic and epigenetic etiology that has only recently been well described. The most prevalent form of the disease, FSHD1, is associated with the contraction of the D4Z4 microsatellite repeat array located on a permissive 4qA chromosome. D4Z4 contraction allows epigenetic derepression of the array, and possibly the surrounding 4q35 region, allowing misexpression of the toxic DUX4 transcription factor encoded within the terminal D4Z4 repeat in skeletal muscles. The less common form of the disease, FSHD2, results from haploinsufficiency of the SMCHD1 gene in individuals carrying a permissive 4qA allele, also leading to the derepression of DUX4, further supporting a central role for DUX4. How DUX4 misexpression contributes to FSHD muscle pathology is a major focus of current investigation. Misexpression of other genes at the 4q35 locus, including FRG1 and FAT1, and unlinked genes, such as SMCHD1, has also been implicated as disease modifiers, leading to several competing disease models. In this review, we describe recent advances in understanding the pathophysiology of FSHD, including the application of MRI as a research and diagnostic tool, the genetic and epigenetic disruptions associated with the disease, and the molecular basis of FSHD. We discuss how these advances are leading to the emergence of new approaches to enable development of FSHD therapeutics. © 2017 American Physiological Society. Compr Physiol 7:1229-1279, 2017.