Resetting the Epigenetic Status of the DM1 Locus

Myotonic dystrophy type 1 is a genetic disorder that causes muscle loss and weakness, affecting a wide range of body functions. The disease is caused by the repeated expansion of the three DNA code letters CTG within the gene dystrophia myotonica protein kinase DMPK. While healthy individuals have only few of these CTG repeats in the DMPK gene, patients having more than 50 of these repeats start showing symptoms. Repeat numbers can reach several thousands, and larger repeat expansions are typically associated with earlier onset and more severe disease. Molecularly, these repeat expansions affect the expression of genes that are important for muscle function. In addition, at the affected genes the repeats also cause changes to the structure of chromatin, the tight complex of DNA and associated proteins. One modification that is known to affect chromatin structure the methylation of DNA, which is often termed an epigenetic mark. Epigenetics is the scientific field that studies heritable changes to chromatin structure and gene expression in the absence of mutations to the DNA sequence. Our collaboration partner Dr. Rachel Eiges at the Shaare Zedek Medical Center in Jerusalem has shown that CTG repeat expansion causes an increase in DNA methylation at certain regions of the DMPK gene. Furthermore, she discovered that removal of the causative repeats in embryonic stem cells reverts also the epigenetic changes at the DMPK gene. In contrast, in muscle progenitor cells the DNA methylation changes persist even when the causative repeats are removed. In a joint project, we are studying the epigenetic mechanisms that upon repeat removal result in the inheritance of DNA methylation in muscle progenitors and in the removal of the marks in embryonic stem cells. My laboratory brings in a long-standing expertise in chromatin biology and epigenomics. In particular, we will develop reporter cells that allow monitoring their epigenetic status. These cells we will then use in screening approaches to identify compounds and genes involved in the maintenance and reversal of chromatin marks following repeat removal. The project thus not only has the potential to uncover novel fundamental mechanisms of epigenetic inheritance but also to contribute novel insights of relevance for future therapeutic approaches.

The project 'Deciphering the mechanisms responsible for resetting the epigenetic status of the DM1 locus in type I myotonic dystrophy' has been conducted in close collaboration between the laboratories of Rachel Eiges (Shaare Zedek Medical Center, Jerusalem, Israel) and Stefan Kubicek (CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria). Thereby, the project benefitted from the combined experise on CTG repeat expansion myotonic dystrophy type I in the Eiges laboratory and on chromatin and chemical biology in the Kubicek laboratory. Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease that affects a wide range of body systems. It results from a CTG repeat expansion (50 - >3,000 triplets) in the 3'-UTR of the dystrophia myotonica protein kinase gene. While DM1 is primarily mediated by RNA/protein gain-of-function mechanisms, it also features local chromatin changes including DNA hypermethylation. However, the developmental timing, kinetics and mechanisms of chromatin alterations in cells from DM1 patients were poorly understood. The Eiges laboratory had discovered an interesting developmentally controlled epigenetic mechanism of locking in chromatin changes at the DM1 locus. While genetic excision of disease-causing repeat expansions in embryonic stem cells fully reverted aberrant chromatin modifications, the same repeat excision in differentated myoblasts is not sufficient to revert these chromatin changes. Understanding this switch has important implications for fundamental questions in epigenetic regulation, but also has the potential to result in improved treatment approaches in the long run. To identify factors that govern this switch from reversible to irreversible DNA methylation, we developed and implemented different assays to directly monitor chromatin changes by measuring DNA methylation with a sequencing readout and to indirectly analyze chromatin structure and epigenetic proteins in flourescent reporter assays. Thereby, we could contribute to discovering a role of the de novo DNA methyltransferases DNMT3A/B in setting the aberrant DNA methylation at the DM1 locus. We further screened for compounds that can alter chromatin structure and localization of chromatin modifying enzymes. We plan to fully validated and characterize the molecular mechanism of action of these compounds in a future follow-up project.