Early de novo DNA methylation and prolonged demethylation in the muscle lineage

Abstract

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Keywords: :

• DNA methylation
• 5-hydroxymethylcytosine
• enhancers
• alternative splicing
• myoblasts
• facioscapulohumeral muscular dystrophy
• histone H3 methylation
• DNaseI hypersensitivity
• muscle
• TET1,

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the ENCODE Chromatin Group at the Broad Institute for the chromatin data, and the ENCODE Transcription Group at California Institute of Technology (Barbara Wold’s group at CalTech for the RNA-seq data and Tom Gingeras’ group at Cold Spring Harbor Laboratory; http://genome.ucsc.edu) for making their data available prepublication. We are very grateful to many FSHD patients and their unaffected relatives without the disease-associated 4q35.2 allele; to Drs V. Vedanarayanan and Rabi Tawil for the muscle samples used to generate myoblast cell strains; and to Dr Stephen Hauschka for the donation of the myosin heavy chain MF20 antibody and advice on culturing conditions for optimal myotube formation. This research was supported by grants from the National Institutes of Health to M.E. (NS04885), R.M.M. (NHGRI 5U54HG004576), G.E.C. (NHGRI U54HG004563), from the FSHD Global Research Foundation to M.E. and by a COBRE grant (NIGMS P20GM103518).