Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells

Shiran Bar,Eran Meshorer,Gal Keshet,Dan Vershkov,Ofra Yanuka,Atilgan Yilmaz,Elyad Lezmi,Naama Meller,Malka Nissim-Rafinia,Nissim Benvenisty,Talia Eldar-Geva

doi:10.1038/s41467-021-26949-7

Abstract

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Highlights

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-oforigin
While extensive loss-of-imprinting occurs in mice following knockout of both Zfp[57] and Znf[445], most imprinted DMRs are still preserved in human embryonic stem cells (ESCs) following knockdown of these genes[22]
We employed a CRISPR/Cas[9] loss-of-function screening to identify factors involved in this process. human parthenogenetic embryonic stem cells (hpESCs) serve as a favorable tool to study imprinting in humans for several reasons: (1) hpESCs consist of only the maternal genome

Summary

Introduction

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-oforigin. It is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Proper expression of imprinted genes is essential for mammalian development, as uniparental embryos having a maternal-only (parthenogenetic) or paternal-only (androgenetic) genomes die during gestation[1,2]. This notion established imprinting as being the block for asexual reproduction in mammals. Exploiting these advantages, we investigated both known and unknown mechanisms that are required for maintaining parental imprinting

Methods

Results

Conclusion