ICF1-Syndrome-Associated DNMT3B Mutations Prevent De Novo Methylation at a Subset of Imprinted Loci during iPSC Reprogramming

Abstract

Parent-of-origin-dependent gene expression of a few hundred human genes is achieved by differential DNA methylation of both parental alleles. This imprinting is required for normal development, and defects in this process lead to human disease. Induced pluripotent stem cells (iPSCs) serve as a valuable tool for in vitro disease modeling. However, a wave of de novo DNA methylation during reprogramming of iPSCs affects DNA methylation, thus limiting their use. The DNA methyltransferase 3B (DNMT3B) gene is highly expressed in human iPSCs; however, whether the hypermethylation of imprinted loci depends on DNMT3B activity has been poorly investigated. To explore the role of DNMT3B in mediating de novo DNA methylation at imprinted DMRs, we utilized iPSCs generated from patients with immunodeficiency, centromeric instability, facial anomalies type I (ICF1) syndrome that harbor biallelic hypomorphic DNMT3B mutations. Using a whole-genome array-based approach, we observed a gain of methylation at several imprinted loci in control iPSCs but not in ICF1 iPSCs compared to their parental fibroblasts. Moreover, in corrected ICF1 iPSCs, which restore DNMT3B enzymatic activity, imprinted DMRs did not acquire control DNA methylation levels, in contrast to the majority of the hypomethylated CpGs in the genome that were rescued in the corrected iPSC clones. Overall, our study indicates that DNMT3B is responsible for de novo methylation of a subset of imprinted DMRs during iPSC reprogramming and suggests that imprinting is unstable during a specific time window of this process, after which the epigenetic state at these regions becomes resistant to perturbation.

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency

“…Our data in pYM-iPSCs support the notion that ZBTB24 is an early factor required for the correct centromeric DNA methylation. We previously generated ICF1 patient-derived iPSCs that are deficient for DNMT3B (38)(39)(40). Comparative mechanistic studies in ICF2 and ICF1 iPSCs will provide insights into the functional interaction between the two proteins and their regulatory pathways.…”

“…The common probes between the arrays were considered using the combineArray function in minfi Bioconductor package v1. 40…”

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency

“…Our data in pYM-iPSCs support the notion that ZBTB24 is an early factor required for the correct centromeric DNA methylation. We previously generated ICF1 patient-derived iPSCs that are deficient for DNMT3B ( 39 – 41 ). Comparative mechanistic studies in ICF2 and ICF1 iPSCs will provide insights into the functional interaction between the two proteins and their regulatory pathways.…”