Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Summary

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.

Graphical abstract

Highlights

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  Integration of CpG-free ssDNA at CGIs induces de novo DNAme in PSCs

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  CpG island methylation response (CIMR) DNA is functional through differentiation

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  CIMR guidelines facilitate genome-wide DNAme editing in pluripotency

Motivation

Pluripotent DNAme edits may be carried through differentiation, alter cell fate, and provide epigenetic models of development and disease, which may be used in future therapeutic DNAMe reversion strategies. To overcome barriers for DNAme editing across entire CpG islands, we investigated whether CpG island methylation responses occur in response to synthetic CpG-free ssDNA integration. We subsequently aimed to optimize CIMR DNAMe editing in pluripotency.

Abstract

Integration of a designer CG-free synthetic ssDNA can stimulate de novo DNAMe at target CGIs. Based on this observation, Tompkins et al. describe a genome-wide DNA methylation editing technique that spreads DNAme across target CpG islands (CGIs), is globally specific, and is functionally maintained through directed differentiation.