Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.

Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of vertebrate and invertebrate embryos. In Drosophila, transduction of the Hedgehog signal is intimately associated with the activity of protein kinase A and the product of the segment polarity gene patched. We have cloned a homologue of patched from the zebrafish Danio rerio and analysed the spatiotemporal regulation of its transcription during embryonic development in both wild-type and mutant animals. We find a striking correlation between the accumulation of patched1 transcripts and cells responding to sonic hedgehog activity both in the neurectoderm and mesoderm, suggesting that like its Drosophila counterpart, patched1 is regulated by sonic hedgehog activity. Consistent with this interpretation, mis-expression of sonic hedgehog results in ectopic activation of patched1 transcription. Using dominant negative and constitutively active forms of the protein kinase A subunits, we also show that expression of patched1 as well as of other sonic hedgehog targets, is regulated by protein kinase A activity. Taken together, our findings suggest that the mechanism of signalling by Hedgehog family proteins has been highly conserved during evolution.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

The expression of the human aldolase A gene is controlled by three alternative promoters. In transgenic mice, pN and pH are active in all tissues whereas pM is activated specifically in adult muscles composed mainly of fast, glycolytic fibers. To detect potential regulatory regions involved in the fast-muscle-specific activation of pM, we analyzed DNase I hypersensitivity in a 4.3-kbp fragment from the 5' end of the human aldolase A gene. Five hypersensitive sites were located near the transcription initiation site of each promoter in those transgenic-mouse tissues in which the corresponding promoter was active. Only one muscle-specific hypersensitive site was detected, mapping near pM. To functionally delimit the elements required for muscle-specific activity of pM, we performed a deletion analysis of the aldolase A 5' region in transgenic mice. Our results show that a 280-bp fragment containing 235 bp of pM proximal upstream sequences together with the noncoding M exon is sufficient for tissue-specific expression of pM. When a putative MEF-2-binding site residing in this proximal pM region is mutated, pM is still active and no change in its tissue specificity is detected. Furthermore, we observed a modulation of pM activity by elements lying further upstream and downstream from pM. Interestingly, pM was expressed in a tissue-specific way in all transgenic mice in which the 280-bp region was present (32 lines and six founder animals). This observation led us to suggest that the proximal pM region contains elements that are able to override to some extent the effects of the surrounding chromatin.