Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion.
Publication Year:
2023
PubMed ID:
37323577
Public Summary:
Although most human cells share the same genetic information, its decoding depends on multiple layers of regulatory mechanisms that influence gene expression. One such layer is the epigenome, the chemical modifications of DNA. In this study, we demonstrate that chemical DNA modifications can be introduced or engineered experimentally to change gene expression. We develop novel tools for DNA modification engineering and gain important insights into the maintenance of the epigenome and regulation of gene expression.
Scientific Abstract:
Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions to regulate transcription factor, chromatin remodeling activity, and genome interpretation. Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs) that stably extends DNAme across target CpG islands (CGIs). Integration of synthetic CpG-free single-stranded DNA (ssDNA) induces a target CpG island methylation response (CIMR) in multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs but not in highly methylated CpG island hypermethylator phenotype (CIMP)+ cancer lines. MLH1 CIMR DNAme spanned the CGI, was precisely maintained through cellular differentiation, suppressed MLH1 expression, and sensitized derived cardiomyocytes and thymic epithelial cells to cisplatin. Guidelines for CIMR editing are provided, and initial CIMR DNAme is characterized at TP53 and ONECUT1 CGIs. Collectively, this resource facilitates CpG island DNAme engineering in pluripotency and the genesis of novel epigenetic models of development and disease.