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

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.

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions

“…We show that ATF7IP and ZMYM2 colocalize together with ZFP57 and SETDB1 at the majority of endogenous ICRs in mESCs and are required for the memory of the epigenetic state at methylated ICRs. This is in line with a publication showing a role of ATF7IP in regulating sperm-specific genes and paternally expressed imprinted genes, including Peg13 in human parthenogenetic ESCs 40 .…”

“…Given their association with H3K9me3 and reported involvement in transcriptional silencing of repetitive elements, we tested their contribution to regulation of epigenetic maintenance at ICRs. In addition human ATF7IP was recently identified to be a repressor of paternally expressed imprinted genes and required for silencing sperm-specific genes 40 . We first wanted to see if these factors indeed localize to the endogenous ICRs and analyzed existing mESC ChIP-seq datasets available for SETDB1 (ref.…”

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development

“…We used published RNA-seq and reduced representation bisulfite-seq (RRBS) data from ZMYM2 -/- hESCs 23,24 to determine if dysregulation of transposons was observed. Indeed, we observed highly significant upregulation of the L1Hs and L1PA2 sub-families, the two youngest LINE element families in humans (Figure 4A).…”

“…ZMYM2 interacts with the Fibronectin III domain of ATF7IP in an apparently SUMO-independent manner, and ATF7IP has been reported to interact with both TRIM28 and the PRC1.6 component MGA 22 . It is striking that loss of either ATF7IP or ZMYM2 results in hypomethylation of young LINE elements in hESCs (Figure 4B,C, Figure S4B-E), and both genes have been hits in CRISPR screens for factors that maintain imprint methylation in stem cells 24,25 . One possibility is that ATF7IP’s primary function at these loci is to recruit ZMYM2 to such elements.…”

“…ZMYM2 was identified in a CRISPR screen as a gene that facilitates exit from pre-implantation-like “naïve” pluripotency in mESCs 21 and has been implicated in transposon silencing in mESCs 16,22 . In subsequent CRISPR screens, ZMYM2 was found to restrict growth in human embryonic stem cells (hESCs) 23 and its deletion results in reactivation of the silenced copy of imprinted genes in both mESCs and hESCs 24,25 . Heterozygous mutations of ZMYM2 in humans cause craniofacial abnormalities and congenital anomalies of the kidney and urinary tract (CAKUT) and Zmym2 +/- mice show elevated rates of CAKUT 15 .…”