# STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

## TL;DR

This study uses STARR-seq to identify enhancers in mouse embryonic stem cells, revealing active, masked, and dormant enhancers that influence cell identity and differentiation.

## Contribution

The study introduces STARR-seq as a method to uncover enhancer activity in mESCs, revealing enhancers that are masked or dormant under standard chromatin assays.

## Key findings

- Active STARR-seq loci show only modest overlap with enhancer locations identified by ChIP-seq.
- ZIC3-bound loci with STARR-seq activity in SL-ESCs are revealed, and Zic3 knockout increases endodermal differentiation.
- Dormant p53-bound enhancers gain activity under specific conditions like Nocodazol treatment or during reprogramming.

## Abstract

Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics.

Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency.

In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.