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: As mentioned before, ChIP-seq experiments upon ectopic DUX4 overexpression in control human muscle cells were previously performed (44). We loaded the ChIP-seq results (GSE33838) on the UCSC genome browser and we found that two DUX4 ChIP-seq peaks were present in the human FRG1 genomic area. The first peak (afterwards referred as FRG1 Peak1) was located inside the second intron of the FRG1 gene, while the second peak (FRG1 Peak2) was located at the 3′ end of the gene (Fig. 2A). Intriguingly, inspection of the ChIP-seq tracks belonging to the ENCODE Enhancer- and Promoter-associated histone marks (50,51) revealed that FRG1 Peak1 (but not FRG1 Peak2) displayed a relative enrichment for Enhancer-associated histone marks (H3K4me1 and H3K27Ac) (Supplementary Material, Fig. S2), suggesting that it might belong to an enhancer regulating FRG1 expression. To verify the DUX4 association to the above regions, we electroporated control human muscle cells with a Myc-tagged form of DUX4 (pCMV-Myc-N-DUX4) or the control pCMV-Myc-N empty vector. Next, we performed chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) using anti-DUX4 or anti-Myc tag antibodies (to support the specificity of the ChIP-qPCR signal derived from DUX4), and with control IgG. As shown in Figure 2B and C, with both anti-Myc and anti-DUX4 antibodies we detected a clear DUX4 enrichment over FRG1 Peak1 and Peak2, with respect to control cells. Intriguingly, while DUX4 enrichment in FRG1 Peak1 was comparable to that of the positive controls RFPL2 and TRIM48 (Fig. 2B and D and E, respectively), FRG1 Peak2 signal was the lowest (Fig. 2C).Figure 2.
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.