This system allows for high-throughput screening of repression of reporter activity and serves as a platform to identify and test therapeutics. of phenotypic similarities between FSHD and an FSHD-like condition caused by FAT1 mutations. Introduction Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders (1) and is characterized by progressive asymmetric skeletal muscle mass weakness that begins in the face, shoulder girdle, and upper arms (2). FSHD-affected individuals also generally exhibit tortuosity of retinal vessels and sensorineural hearing loss (3,4). The causative genetic defect is the loss of transcriptional repression of the Double Homeobox Protein 4 (DUX4) gene present in each repeat of the macrosatellite array (D4Z4) at chromosome 4q35 (5C7). Chromatin is usually relaxed as a result Enecadin of array contraction to <11 repeats (FSHD1) (8) or mutation of epigenetic modifiers of the region (FSHD2) (9) and results in aberrant expression of DUX4 in muscle mass cells (10C12). Controlling the pathogenesis of FSHD by halting or reversing its progression will require a detailed understanding of the events that occur downstream of DUX4 activation. The forced expression of DUX4 using strong viral promoters in cultured cells prospects to aberrant activation of a cascade of diverse targets and produces transcripts from normally dormant transposable elements, transcripts characteristic of an innate immune response and germline-specific transcripts normally silenced in muscle mass cells (13). Exogenous expression of DUX4 is also harmful to cultured mouse myoblasts (14), disrupts Xenopus development (15) and results in p53-dependent muscle damage in adult mice and zebrafish (16). Germline expression in mice results in embryonic lethality and/or runting and produces a unique skin phenotype due to failure of basal keratinocyte migration. These mice also exhibit retinal vessel tortuosity reminiscent of that found in FSHD (17). While much has been learned from analyzing cells where DUX4 has been forcibly expressed, the pattern and level of endogenous DUX4 expression in FSHD myoblasts are substantially more delicate. DUX4 transcripts are found in a small percentage of cultured mononuclear FSHD myoblasts that appear to grow and divide without an obvious phenotype (18). Recently, we exhibited that sporadic DUX4 expression occurs almost exclusively in differentiated FSHD myotubes. When myoblast fusion is usually extensive, DUX4 protein can be detected in many myonuclei and cultures of FSHD myotubes demonstrate DUX4-mediated cytotoxicity, even when only a portion of nuclei are actively transcribing DUX4 (19). Importantly, we Enecadin fused human FSHD myoblasts with mouse C2C12 cells to Enecadin demonstrate that DUX4 expression from a single nucleus can result in diffusion of DUX4 protein to adjacent nuclei within the same myotube. The spatial and temporal relationship between DUX4 expression and the induction of transcription from DUX4 target genes is usually a less analyzed but important feature of DUX4-mediated cytotoxicity. Several groups have correlated marks of apoptosis with DUX4 expression, including events resulting from forced expression in adult mouse Hyal1 muscle mass (16), human cell lines (15) or from presumably endogenous DUX4 expression in FSHD muscle tissue (20). TUNEL-positive foci exist in human FSHD myotube cultures but do not co-localize with DUX4 immunofluorescence, suggesting that apoptosis may only occur when DUX4 is usually expressed at very high supraphysiologic levels, or that apoptosis is usually temporally disconnected from DUX4 protein in FSHD myotubes. Supporting the latter, we found that the treatment with anti-apoptotic chemicals could prevent death seen in FSHD myotube cultures (21). This obtaining led us to hypothesize that this expression of DUX4 is usually momentary, though impactful enough to leave a lasting and detrimental transcriptional signature that results in muscle mass death. Forced overexpression of DUX4 could cause molecular signatures that may be unrelated to FSHD. Given the.
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