Direct interplay between two candidate genes in FSHD muscular dystrophy.
Bottom Line: The major form of the disease (FSHD1) is linked to decrease in copy number of a 3.3-kb tandem repeated macrosatellite (D4Z4), located on chromosome 4q35.We found also that ectopically expressed DUX4 up-regulates the endogenous human FRG1 gene in healthy muscle cells, while DUX4 knockdown leads to a decrease in FRG1 expression in FSHD muscle cells.Intriguingly, the mouse Frg1 genomic area lacks DUX4 binding sites and DUX4 is unable to activate the endogenous mouse Frg1 gene providing a possible explanation for the lack of muscle phenotype in DUX4 transgenic mice.
Affiliation: Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Dulbecco Telethon Institute at San Raffaele Scientific Institute, DIBIT2, 5A3, Via Olgettina 58, 20132 Milan, Italy Università Vita-Salute San Raffaele, Milan, Italy.Show MeSH
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Mentions: To investigate whether DUX4 might function as a FRG1 transcriptional activator through the identified genomic regions, we cloned FRG1 Peak1 or Peak2 regions upstream of the Firefly Luciferase reporter gene (Fig. 3A). We co-transfected the constructs together with pCIneo-DUX4 expression vector or with the corresponding pCIneo empty vector, and with a Renilla Luciferase expression vector to normalize the results. Surprisingly, we found that DUX4 was able to transactivate selectively FRG1 Peak1 region, but not FRG1 Peak2 region (Fig. 3B). Interestingly, inspection of the nucleotide sequence underlying FRG1 Peak regions revealed that the previously described DUX4 consensus binding site (TAAYBBAATCA, IUPAC nomenclature) (44) was present in FRG1 Peak1 (TAATTCAATCA), while FRG1 Peak2 displayed only a partial sequence (TAATGTA), providing a plausible explanation for the differential activity of the two regions. To investigate the functional relevance of our findings, we mutated the DUX4 core motif present in FRG1 Peak1 region. Mutations of FRG1 Peak1 region (Fig. 3A) ablated the transactivation mediated by DUX4 (Fig. 3B) supporting the sequence-specificity of DUX4-mediated transcriptional activation of FRG1 gene expression.Figure 3.
Affiliation: Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Dulbecco Telethon Institute at San Raffaele Scientific Institute, DIBIT2, 5A3, Via Olgettina 58, 20132 Milan, Italy Università Vita-Salute San Raffaele, Milan, Italy.