Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus.

Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.

The Contribution of Pluripotent Stem Cell (PSC)-Based Models to the Study of Fragile X Syndrome (FXS).

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a deficiency in the fragile X mental retardation protein (FMRP) due to a CGG repeat expansion in the 5'-UTR of the X-linked FMR1 gene. When CGGs expand beyond 200 copies, they lead to epigenetic gene silencing of the gene. In addition, the greater the allele size, the more likely it will become unstable and exhibit mosaicism for expansion size between and within tissues in affected individuals. The timing and mechanisms of FMR1 epigenetic gene silencing and repeat instability are far from being understood given the lack of appropriate cellular and animal models that can fully recapitulate the molecular features characteristic of the disease pathogenesis in humans. This review summarizes the data collected to date from mutant human embryonic stem cells, induced pluripotent stem cells, and hybrid fusions, and discusses their contribution to the investigation of FXS, their key limitations, and future prospects.

The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA.

Pathological mutations involving noncoding microsatellite repeats are typically located near promoters in CpG islands and are coupled with extensive repeat instability when sufficiently long. What causes these regions to be prone to repeat instability is not fully understood. There is a general consensus that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures composed of persistent DNA:RNA hybrids, concomitant with single-strand DNA displacements (R-loops). Using human embryonic stem cells with wild-type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute preferential sites (hotspots) for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double-strand structures by the nontranscribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats considerably expand. Further, we demonstrate that when the CGGs in FMR1 are hyper-methylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step toward the identification of dynamic, unconventional DNA structures across the G-rich repeats at FMR1 and C9orf72 disease-associated loci.