Search

Facioscapulohumeral dystrophy (FSHD; OMIM #158900, #158901) is caused by mis-expression of the DUX4 transcription factor in skeletal muscle1. Animal models of FSHD are hampered by incomplete knowledge of the conservation of the DUX4 transcriptional program in other species2–5. Despite divergence of their binding motifs, both mouse Dux and human DUX4 activate genes associated with cleavage-stage embryos, including MERV-L and ERVL-MaLR retrotransposons, in mouse and human muscle cells respectively. When expressed in mouse cells, human DUX4 maintained modest activation of cleavage-stage genes driven by conventional promoters, but did not activate MERV-L-promoted genes. These findings indicate that the ancestral DUX4-factor regulated genes characteristic of cleavage-stage embryos driven by conventional promoters, whereas divergence of the DUX4/Dux homeodomains correlates with retrotransposon specificity. These results provide insight into how species balance conservation of a core transcriptional program with innovation at retrotransposon promoters and provide a basis for animal models that recreate the FSHD transcriptome.

Background Facioscapulohumeral dystrophy (FSHD) is a progressive muscle disease caused by mutations that lead to epigenetic derepression and inappropriate transcription of the double homeobox 4 (DUX4) gene in skeletal muscle. Drugs that enhance the repression of DUX4 and prevent its expression in skeletal muscle cells therefore represent candidate therapies for FSHD. Methods We screened an aggregated chemical library enriched for compounds with epigenetic activities and the Pharmakon 1600 library composed of compounds that have reached clinical testing to identify molecules that decrease DUX4 expression as monitored by the levels of DUX4 target genes in FSHD patient-derived skeletal muscle cell cultures. Results Our screens identified several classes of molecules that include inhibitors of the bromodomain and extra-terminal (BET) family of proteins and agonists of the beta-2 adrenergic receptor. Further studies showed that compounds from these two classes suppress the expression of DUX4 messenger RNA (mRNA) by blocking the activity of bromodomain-containing protein 4 (BRD4) or by increasing cyclic adenosine monophosphate (cAMP) levels, respectively. Conclusions These data uncover pathways involved in the regulation of DUX4 expression in somatic cells, provide potential candidate classes of compounds for FSHD therapeutic development, and create an important opportunity for mechanistic studies that may uncover additional therapeutic targets. Electronic supplementary material The online version of this article (doi:10.1186/s13395-017-0134-x) contains supplementary material, which is available to authorized users.

Facioscapulohumeral dystrophy (FSHD) is caused by the mis-expression of DUX4 in skeletal muscle cells. DUX4 is a transcription factor that activates genes normally associated with stem cell biology and its mis-expression in FSHD cells results in apoptosis. To identify genes and pathways necessary for DUX4-mediated apoptosis, we performed an siRNA screen in an RD rhabdomyosarcoma cell line with an inducible DUX4 transgene. Our screen identified components of the MYC-mediated apoptotic pathway and the double-stranded RNA (dsRNA) innate immune response pathway as mediators of DUX4-induced apoptosis. Further investigation revealed that DUX4 expression led to increased MYC mRNA, accumulation of nuclear dsRNA foci, and activation of the dsRNA response pathway in both RD cells and human myoblasts. Nuclear dsRNA foci were associated with aggregation of the exon junction complex component EIF4A3. The elevation of MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates in FSHD muscle cells suggest that these processes might contribute to FSHD pathophysiology., Author summary Facioscapulohumeral dystrophy (FSHD) is a common form of muscular dystrophy which is currently untreatable. It is caused by the inappropriate expression in skeletal muscle of the gene DUX4 that encodes a transcription factor normally expressed in some stem cells. When DUX4 is expressed in cultured human or mouse skeletal muscle cells, it activates a program of cell death. Knowing the molecular basis for the cell death induced by DUX4 is important to determine the mechanism of muscle damage in FSHD. We used a molecular screening approach to identify genes and pathways necessary for DUX4 to induce the cell death program. We found that DUX4 activated a known MYC-induced cell death pathway, at least in part through stabilization of MYC mRNA. We also found that DUX4 expression led to an accumulation of double stranded RNAs (dsRNAs) that induced a cell death pathway evolved to protect against viral infections. This dsRNA accumulation was accompanied by aggregation of the EIF4A3 protein, a factor involved in mRNA surveillance and decay, which may provide a partial mechanism for how DUX4 can inhibit RNA quality control pathways in cells. Because FSHD muscle cells have increased MYC mRNA, dsRNA accumulation, and EIF4A3 nuclear aggregates, we conclude that these processes might contribute to FSHD pathophysiology.

A new class pyridinium-based liquid crystals with an extended fluorinated pyrrolidine were synthesized. These compounds show a wide mesophase range and are stable to high temperatures after an introduction of a fluorinated pyrrolidine to the terminal end. Their properties were modified by varying alkyl substituents on the pyridinium ring. The mesophase behavior was affected by altering the configuration of the terminal fluorinated heterocycle. For a comparison, the analogs 5a–5c with an extended fluorinated azepine were also synthesized. The terminal seven-membered 3,3,4,4,5,5,6,6-octafluoroazepane formed an “L” shape. The twisted L configuration significantly impacted the liquid crystal behavior of 5a–5c, resulting from steric hindrance. The structures of 3b were investigated using single crystal X-ray diffraction analysis.

SummaryMost transcription factor families contain highly related paralogs generated by gene duplication, and functional divergence is generally accomplished by activation of distinct sets of genes by each member. Here we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find that MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits Pol II, and robustly activates gene transcription. Therefore, the initial specification of the muscle lineage by Myf5 occurs without significant induction of gene transcription. Transcription of the skeletal muscle program is then achieved by the subsequent expression of MyoD, which binds to the same sites as Myf5, indicating that each factor regulates distinct steps in gene initiation and transcription at a shared set of binding sites.

SummaryMyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG) and a “private” sequence (CAGGTG or CAGATG, respectively). To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

In the title fluorinated pyrrolidine derivative, C10H10F4N2, the dihedral angle between the best planes of the benzene and pyrrolidine rings is 62.6 (1)°. The crystal packing features intermolecular N—H...F hydrogen bonds.

The asymmetric unit of the title compound, C14H12F8N2, contains one tetrafluoropyrrolidine system and one half-molecule of benzene; the latter, together with a second heterocyclic unit, are completed by symmetry, with a twofold crystallographic axis crossing through both the middle of the bond between the C atoms bearing the heterocyclic rings and the opposite C—C bonds of the whole benzene molecule. The pyrrolidine ring shows an envelope conformation with the apex at the N atom. The dihedral angle between the least-squares plane of this ring and the benzene ring is 36.9 (5)°. There are intramolecular C—H...N interactions generating S(6) ring motifs. In the crystal structure, the molecules are linked by C—H...F interactions, forming chains parallel to [010].

In the title compound, C11H8F4O3, the eight-membered dialkoxy ring adopts a highly puckered conformation. In the crystal, molecules are linked by weak C—H...O interactions.

Summary The regulatory networks of differentiation programs have been partly characterized; however, the molecular mechanisms of lineage-specific gene regulation by highly similar transcription factors remain largely unknown. Here we compare the genome-wide binding and transcription profiles of NEUROD2-mediated neurogenesis with MYOD-mediated myogenesis. We demonstrate that NEUROD2 and MYOD bind a shared CAGCTG E box motif and E box motifs specific for each factor: CAGGTG for MYOD and CAGATG for NEUROD2. Binding at factor-specific motifs is associated with gene transcription, whereas binding at shared sites is associated with regional epigenetic modifications but is not as strongly associated with gene transcription. Binding is largely constrained to E boxes preset in an accessible chromatin context that determines the set of target genes activated in each cell type. These findings demonstrate that the differentiation program is genetically determined by E box sequence, whereas cell lineage epigenetically determines the availability of E boxes for each differentiation program.