# Coordinated repression of totipotency-associated gene loci by histone methyltransferase EHMT2 via LINE1 regulatory elements

## TL;DR

This study shows how the protein EHMT2 prevents mouse stem cells from becoming either more specialized or reverting to a more primitive state by controlling gene activity.

## Contribution

The paper identifies EHMT2 as a key regulator of bidirectional differentiation in mouse pluripotent cells through distinct repression mechanisms.

## Key findings

- EHMT2 represses ZGA-associated genes in clusters called ECORDs by spreading H3K9me2 from LINE-1 elements.
- EHMT2 inhibits DPPA2/4 activation at ERVs and works with ZFP462 to repress germ layer genes.
- Depleting EHMT2 increases ZGA gene expression and promotes the mESC-to-2CLC transition.

## Abstract

Mouse embryonic stem cells (mESCs), in addition to differentiating into the three germ layers, can reverse typical developmental trajectories, as exemplified by their ability to de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). This unique property offers the opportunity to elucidate the molecular principles that govern the pre-implantation stages of mammalian development. Here, we dissect the functions of the chromatin repressor EHMT2, a candidate antagonist of the mESC-to-2CLC transition, by leveraging a multipurpose allele for acute protein depletion and efficient immunoprecipitation. Our experiments revealed distinct principles of EHMT2-mediated gene repression in mESCs based on specific chromatin binding patterns and protein co-factors. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. EHMT2 counteracts the recruitment of the activator DPPA2/4 to promoter-proximal endogenous retroviral elements (ERVs) at 2CLC genes. EHMT2 depletion enhances the expression of ZGA-associated transcripts in 2CLCs and synergizes with spliceosome inhibition and retinoic acid signaling to facilitate the mESC-to-2CLC transition. In contrast to ZGA-associated genes, the repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters, in collaboration with ZFP462, and involves binding to non-repetitive candidate enhancers. Our observations provide novel mechanistic insight into how pluripotent cells achieve attenuation of their bidirectional differentiation potential and reveal unique transcriptional features of murine totipotent cells.

The histone methyltransferase EHMT2 employs distinct molecular mechanisms to counteract both forward (into germ layers) and backward (into totipotent-like cells) differentiation in mouse pluripotent cells.

Acute EHMT2 depletion in mouse ESCs results in the reactivation of nearly 100 clusters of co-regulated ZGA-associated genes, which we coin “EHMT2 coordinately repressed domains” (ECORDs).EHMT2 in ECORDs binds to evolutionarily young LINE-1 elements, which serve as docking sites for both chromatin repressors and activators.EHMT2 counteracts DPPA2/4-mediated gene activation in ECORDs and synergizes with ZFP462 to repress germ layer-associated genes.

Acute EHMT2 depletion in mouse ESCs results in the reactivation of nearly 100 clusters of co-regulated ZGA-associated genes, which we coin “EHMT2 coordinately repressed domains” (ECORDs).

EHMT2 in ECORDs binds to evolutionarily young LINE-1 elements, which serve as docking sites for both chromatin repressors and activators.

EHMT2 counteracts DPPA2/4-mediated gene activation in ECORDs and synergizes with ZFP462 to repress germ layer-associated genes.

The histone methyltransferase EHMT2 employs distinct molecular mechanisms to counteract both forward (into germ layers) and backward (into totipotent-like cells) differentiation in mouse pluripotent cells.