Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq.

Laura V Glaser,Sebastiaan H Meijsing,Ho-Ryun Chung,Mara Steiger,Alena Van Bömmel,Alisa Fuchs,Martin Vingron,Edda Einfeldt

doi:10.1093/nar/gkab1100

Abstract

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.

Highlights

Gene expression in eukaryotic cells is a tightly regulated process which is a prerequisite for cellular identity
To limit the complexity of the library, we prioritized regions that are likely to act as enhancers [52] by focusing on accessible chromatin isolated by FAIRE as input material for our STARR-seq [38] library (Figure 1A–C)
The introduction of unique molecular identifiers (UMIs) allows one to distinguish between independent transcript replicates and PCR duplicates that can dramatically distort the relative quantities of individual fragments within a library [54]

Summary

Introduction

Gene expression in eukaryotic cells is a tightly regulated process which is a prerequisite for cellular identity. Regulation of transcription is controlled by transcription factors (TF) and the regulatory genomic elements (enhancers, promoters) they target [1,2]. The selective and combinatorial activation of enhancers in a spatiotemporal manner allows for the complexity of higher eukaryotic organisms, which consist of a large number of different highly specialized cells they all possess the same genome [3,4]. Enhancers are defined as the genomic elements that can control the activity of promoters whereas promoters are the regions where transcription of genes is initiated. The assignment of enhancers to their target promoters is an important step in elucidating gene regulation and has been addressed in recent years with rapidly evolving high-throughput chromatin interaction assays [10,11,12]. The functional relevance of identified enhancer-promoter pairs was mainly investigated for individual genes or loci [13,14,15,16,17,18,19] and remains a largely unsolved problem at the genome-wide level

Methods

Results

Conclusion