Molecular Roles of Double Homeobox 4 in Facioscapulohumeral Muscular Dystrophy
Open AccessFacioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular dystrophy. The leading hypothesis of FSHD pathogenesis is that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat at chromosome 4q35 causes the disease. The overall goal of this study was to identify the upstream transcriptional regulators and downstream transcriptional targets of the DUX4 gene and to advance our understanding of the molecular mechanisms of FSHD. In chapter 1, the DNA pulldown assay was performed using a 282 b.p. biotin labeled DUX4 promoter probe coupled with LTQ mass spectrometry to identify proteins that interact with the promoter region of the DUX4 gene. Among a total of 13 proteins specifically pulled down by the DUX4 probe, poly (ADP-ribose) polymerase 1 (PARP1) was ranked the highest by the number of peptides detected. The result was validated by DNA pulldown assay coupled with immunoblotting (2-fold enrichment, p<0.05). A chromatin immunoprecipitation (ChIP) assay was then performed and showed a 65-fold enrichment (p<0.01) of the DUX4 promoter fragment in immortalized FSHD myoblasts, while no enrichment was observed in the myoblasts of an unaffected sibling. PARP1 has been shown to physically interact with DNA methyltransferase 1 (DNMT1) and suppress DNMT1 activity by poly-ADP ribosylation. To determine whether DNMT1 was also enriched at the DUX4 promoter region, the DNA pulldown assay coupled with immunoblotting was performed and showed a 2-fold enrichment (p<0.01) of DNMT1. The results suggest that PARP1 and DNMT1 bind the promoter region of the DUX4 gene, which may contribute to the hypomethylation of the region and potentially affect the expression of the DUX4 gene in FSHD. In chapter 2, transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage were determined by genome-wide expression profiling. Human rhabdomyosarcoma (RD) and mouse C2C12 cells transfected with expression vectors encoding DUX4 were profiled using Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. Among the 2267 and 150 transcripts differentially expressed in the RD and C2C12 cells, respectively, myogenic differentiation1 (MYOD), myogenin (MYOG) and six MYOD downstream targets were upregulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells. Major molecular pathways were differentially affected in the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Among the 40 transcripts regulated by DUX4 in both the RD and C2C12 cells, urotensin 2 (UTS2) was significantly induced in the RD (76-fold, p<0.01) and C2C12 cells (224 fold, p<0.05), respectively. Using real-time quantitative RT-PCR, I further showed that MYOD (19-fold, p<0.01), MYOG (110-fold, p<0.01) and UTS2 (229-fold, p<0.01) were upregulated in immortalized FSHD myoblasts. The findings suggest that DUX4 differentially regulates mouse and human transcriptomes, therefore mouse cells and models may not be suitable for studying molecular mechanisms of FSHD. In chapter 3, ChIP assays were performed to determine whether DUX4 interacts with the promoters of MYOD and UTS2 directly. ChIP assays were performed to detect direct interaction between the DUX4 and the promoters of UTS2 and MYOD. The DUX4 was ectopically expressed in RD cells using either pCIneo-DUX4 or FLAG-DUX4 vectors. The results showed no PCR amplification using primers targeting the promoter fragments of MYOD, UTS2 nor a positive control, paired-like homeodomain 1 (PITX1), after the ChIP was performed. I concluded that this was likely due to technical issues associated with the ChIP assays performed. We then performed luciferase reporter assay to investigate the function of Uts2. Luciferase reporter assay using the pGL3basic vector containing a 271 b.p. promoter sequence of the Uts2 was co-transfected with a DUX4 expression vector. The results showed that ectopically expressing DUX4 activated the luciferase reporter gene driven by the Uts2 promoter (467-fold, p<0.01). Based on findings of this project, I propose a hypothesized disease model wherein PARP1 binds the DUX4 promoter and inhibits the catalytic activity of DNMT1 in FSHD muscle cells. This leads to hypomethylation of the DUX4 promoter region and subsequent derepression of the DUX4 gene. DUX4 can then activate genes involved in oxidative stress response, and myogenesis, which contribute to the FSHD.
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