GSE128901
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. Focusing on myotonic dystrophy type 1 (DM1), we discovered a fundamental difference between undifferentiated and differentiated cells. While in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (2000CTG), in patients' myoblasts (CTG2600 expansion) repeat deletion fails to do so. This distinction stems from cell differentiation, and can be set back by reprogramming 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 and/or DNMT3a). Overall, these findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus. Overall design: Total RNA was extracted using NucleoSpin RNA Plus kit (Macherey-Nagel, Cat # 740984.50) following the manufacturer's instructions. RNA was quantitated by NanoDrop 200 (ThermoFisher). RNA libraries were prepared for sequencing using standard Illumina protocols. Genomic DNA (2 µg) was modified by bisulfite treatment (EZ DNA methylation kit, Zymo Research) and amplified by FastStart DNA polymerase (Roche). PCR was carried out using 5? primer: 5?-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGTTGTGGGTTAGTGTT-3?, and 3? primer: 5?-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCAACAACCTACAACTATTAT-3? (DMPK-specific sequences are underlined; adaptors for library preparation are at the 5? end of each primer). Amplified products were quality controlled for size using the D1000 ScreenTape kit; Agilent Technologies) and for concentration using the Qubit® DNA HS Assay kit (catalog #32854; Invitrogen). Subsequently, the PCR products were bead purified with Agencourt Ampure XP beads (Beckman Coulter) according to the manufacturer?s protocol. Next, amplicons were subjected to a second PrimMax Takara PCR reaction (1st purified PCR DNA (7.5 ?l), 2.5 ?l N7XX primer (nextera barcode 1), 2.5 ul S5XX primer (nextera barcode 2), 12.5 ?l 2x Primstar ReadyMix). PCR program: 98°C for 1 minute followed by 8 cycles of 98°C for 10 seconds, 55°C for 10 seconds, 72°C for 30 seconds, then 72°C for 5 minutes and finally held at 10°C. The 2nd PCR was bead purified, quality controlled using the D1000 ScreenTape kit and the Qubit® DNA HS Assay kit, and all resultant libraries were normalized and pooled at a 10 nM concentration. Denaturation and dilution of the sequencing pool were performed according to the standard Illumina protocol. Ultimately, 1.5 pM of pool (combined with 40% spiked?in) was loaded onto a NextSeq 500 Mid?Output Kit (150 cycles) cartridge (catalog #FC?102?1001; Illumina, San Diego, CA) for high throughput sequencing on a NextSeq 500 instrument (Illumina), with 150 cycle, single?read run conditions.