McNamara, T. F. | bioRxiv Science

bioRxiv2020

A susceptibility locus on chromosome 13 profoundly impacts the stability of genomic imprinting in mouse pluripotent stem cells

Cultured pluripotent cells accumulate detrimental epigenetic alterations, including DNA methylation changes at imprinted genes known as loss-of-imprinting (LOI). Despite the substantial biomedical relevance of this phenomenon, the molecular cause of this epigenetic instability in pluripotent cells remains unknown. While the occurrence of LOI is generally considered a stochastic phenomenon, here we document a strong genetic determinant that segregates mouse pluripotent cells into epigenetically stable and unstable cell lines. Unstable lines exhibit hypermethylation at Dlk1-Dio3 and select other imprinted loci, which is associated with impaired developmental potential. Stimulation of demethylases by ascorbic acid prevents LOI and can preserve developmental potential. Susceptibility to LOI greatly differs between commonly used mouse strains, which we utilize to map a causal region on chromosome 13 with Quantitative Trait Locus (QTL) analysis. Our observations identify a strong genetic determinant of locus-specific epigenetic abnormalities in pluripotent cells and provide a non-invasive way to suppress them. This highlights the importance of considering genetics in conjunction with culture conditions for assuring the quality of pluripotent cells for biomedical applications.

cell biology↗

bioRxiv2019

Chromosomal rearrangements at hypomethylated Satellite 2 sequences are associated with impaired replication efficiency and increased fork stalling

Cancer cells, aging cells, and cells from patients with the developmental disorder Immunodeficiency, Centromeric instability, and Facial anomalies (ICF) syndrome frequently display a striking loss of DNA methylation (hypomethylation) that is accompanied by increased DNA damage and chromosomal rearrangements. Despite the robust link, the mechanism by which hypomethylation leads to genomic instability is poorly understood. We report that the human pericentromeric repeat sequence Satellite 2 (SAT2) poses challenges to the DNA replication machinery when hypomethylated. Loss of methylation at SAT2 is associated with increased frequencies of chromosomal abnormalities and DNA damage. Hypomethylation of SAT2 is associated with elevated levels of replication stress signaling, and chromosomal abnormalities involving SAT2 are enhanced by low levels of aphidicolin-induced replication stress. To investigate the basis for these chromosomal abnormalities, we developed a single-molecule approach employing DNA combing to examine the progress of replication forks through SAT2 at the resolution of a single DNA molecule. Our analysis of replicating SAT2 molecules provides in vivo evidence that hypomethylation of SAT2 strongly decreases the efficiency of replicating these sequences suggesting that hypomethylation results in the formation of barriers to the replication machinery. Consistent with increased frequency of fork stalling at these sequences, we find increased levels of single-stranded DNA (ssDNA) binding protein RPA2 as well as asymmetric progression of sister replication forks within hypomethylated SAT2 sequences. Together these findings indicate that impaired replication triggers the formation of chromosomal aberrations observed at hypomethylated SAT2 sequences and also suggests a mechanistic basis for how the loss of DNA methylation may contribute to genomic instability in diverse pathological conditions.

molecular biology↗