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Direct interplay between two candidate genes in FSHD muscular dystrophy.

Ferri G, Huichalaf CH, Caccia R, Gabellini D - Hum. Mol. Genet. (2014)

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.

View Article: PubMed Central - PubMed

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.

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FRG1 Peak1 region is sufficient for specific DUX4 transactivation. (A) Schematic representation of the constructs employed in luciferase assays. Genomic fragments of the FRG1 gene containing DUX4 putative binding sites were cloned upstream of the SV40 promoter, which controlled the expression of the Luciferase reporter gene. DUX4 core binding motifs within FRG1 genomic regions are reported. The three nucleotides of FRG1 Peak1 region that were mutated to generate FRG1 Peak1-mutated region are underlined. (B) FRG1 Peak1 region is able to transactivate the Luciferase reporter gene in the presence of DUX4, while the mutation in key DUX4 consensus sequence nucleotides abolishes the transactivation. DUX4 is not able to transactivate the Luciferase gene through FRG1 Peak2 region (paired t-test, *P < 0.05, n = 3, mean ± SEM).
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DDU536F3: FRG1 Peak1 region is sufficient for specific DUX4 transactivation. (A) Schematic representation of the constructs employed in luciferase assays. Genomic fragments of the FRG1 gene containing DUX4 putative binding sites were cloned upstream of the SV40 promoter, which controlled the expression of the Luciferase reporter gene. DUX4 core binding motifs within FRG1 genomic regions are reported. The three nucleotides of FRG1 Peak1 region that were mutated to generate FRG1 Peak1-mutated region are underlined. (B) FRG1 Peak1 region is able to transactivate the Luciferase reporter gene in the presence of DUX4, while the mutation in key DUX4 consensus sequence nucleotides abolishes the transactivation. DUX4 is not able to transactivate the Luciferase gene through FRG1 Peak2 region (paired t-test, *P < 0.05, n = 3, mean ± SEM).

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.


Direct interplay between two candidate genes in FSHD muscular dystrophy.

Ferri G, Huichalaf CH, Caccia R, Gabellini D - Hum. Mol. Genet. (2014)

FRG1 Peak1 region is sufficient for specific DUX4 transactivation. (A) Schematic representation of the constructs employed in luciferase assays. Genomic fragments of the FRG1 gene containing DUX4 putative binding sites were cloned upstream of the SV40 promoter, which controlled the expression of the Luciferase reporter gene. DUX4 core binding motifs within FRG1 genomic regions are reported. The three nucleotides of FRG1 Peak1 region that were mutated to generate FRG1 Peak1-mutated region are underlined. (B) FRG1 Peak1 region is able to transactivate the Luciferase reporter gene in the presence of DUX4, while the mutation in key DUX4 consensus sequence nucleotides abolishes the transactivation. DUX4 is not able to transactivate the Luciferase gene through FRG1 Peak2 region (paired t-test, *P < 0.05, n = 3, mean ± SEM).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321439&req=5

DDU536F3: FRG1 Peak1 region is sufficient for specific DUX4 transactivation. (A) Schematic representation of the constructs employed in luciferase assays. Genomic fragments of the FRG1 gene containing DUX4 putative binding sites were cloned upstream of the SV40 promoter, which controlled the expression of the Luciferase reporter gene. DUX4 core binding motifs within FRG1 genomic regions are reported. The three nucleotides of FRG1 Peak1 region that were mutated to generate FRG1 Peak1-mutated region are underlined. (B) FRG1 Peak1 region is able to transactivate the Luciferase reporter gene in the presence of DUX4, while the mutation in key DUX4 consensus sequence nucleotides abolishes the transactivation. DUX4 is not able to transactivate the Luciferase gene through FRG1 Peak2 region (paired t-test, *P < 0.05, n = 3, mean ± SEM).
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.

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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus