STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells
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RESEARCH
Open Access
STARR-seq identifies active, chromatinmasked, and dormant enhancers in pluripotent mouse embryonic stem cells Tianran Peng1, Yanan Zhai1,2,3, Yaser Atlasi1, Menno ter Huurne1, Hendrik Marks1, Hendrik G. Stunnenberg1,2*† and Wout Megchelenbrink1,2,3*† * Correspondence: H.Stunnenberg@ prinsesmaximacentrum.nl; W.L. Megchelenbrink@ prinsesmaximacentrum.nl † Hendrik G. Stunnenberg and Wout Megchelenbrink contributed equally to this work. 1 Department of Molecular Biology, Radboud Institute for Molecular Life Sciences, Radboud University, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands Full list of author information is available at the end of the article
Abstract Background: 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. Results: 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. Conclusions: 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.
Introduction Mouse embryonic stem cells (mESCs) derived from the inner cell mass of the early developing embryo can propagate indefinitely in vitro and are pluripotent [1–3]. Pluripotent stem cells can give rise to all somatic cell lineages, a fundamental property for the development of complex organisms, including humans, that holds great promise for regenerative medicine [4–6]. Murine ESCs cultured in medium supplemented with serum and leukemia inhibitory factor (serum + LIF; SL) are metastable and prone to spontaneous © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
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