Epigenetic analysis of the DMD locus

The Dystrophin gene (DMD) is the largest gene in the human genome, mapping on Xp21, spanning 2.2Mb and accounting for approximately 1% of the entire human genome. Mutations in this gene cause Duchenne and Becker muscular dystrophy, X-linked dilated cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 hours, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA pol II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. More importantly, epigenetic marks highlighted the existence of four novel cis-DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies. ChIP/chip on DMD locus in SJCRH30 and HeLa cell line using Anti-RNA Poyimerase II, Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2),Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2), Anti-H3ac and Anti-H3K4me2. Experimental duplicates were provided for both cell lines and for all antibodies with exception of Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5), Anti-H3ac and Anti-H3K4me2 in HeLa cells.