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

Abstract

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

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

“…Embryonic stem cells adapted to continuous pluripotent culture may be more resistant to differentiation than developing mice. We also do not observe loss of canonical imprints in Zmym2 -/- embryos 24,25 (Figure S3I). It may be that the dynamic state of mESCs, in which both de novo methylation and demethylation occur in continuous culture, does not reflect methylation dynamics in the rapidly developing mouse embryo.…”

“…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 .…”

Genomic context sensitizes regulatory elements to genetic disruption

“…While stable parent-of-origin specific methylation is established at the Igf2 / H19 ICR during development, the methylation pattern maintained in cultured mESCs is more labile (Swanzey et al 2020). In addition, controlling methylation status of transfected DNA would require additional steps (Schübeler et al 2000; Butz et al 2022). Thus we developed a genetic approach to reliably direct CTCF occupancy at the ICR ( Figure 1d ).…”

Loss of CpG island immunity to DNA methylation induced by mutation

“…There is a precedent where naturally acquired DNA methylation survives the global demethylation that accompanies early mammalian development (although the modification is erased prior to the next generation). This concerns imprinted genes, where the methylated alleles specifically recruit KRAB zinc finger proteins which, with the aid of the repressor KAP1, protect the methylated allele [ 7 ]. Persistent methylation of transposable elements can also involve KRAB zinc finger proteins, but in this case DNA methylation is transiently lost during differentiation of the germline and subsequently restored as development proceeds [ 8 ].…”