Abstract
Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator Sox2 and its essential enhancer Sox2 Control Region (SCR) in living embryonic stem cells (ESCs). We find that Sox2 and SCR show no evidence of enhanced spatial proximity and that spatial dynamics of this pair is limited over tens of minutes. Sox2 transcription occurs in short, intermittent bursts in ESCs and, intriguingly, we find this activity demonstrates no association with enhancer proximity, suggesting that direct enhancer-promoter contacts do not drive contemporaneous Sox2 transcription. Our study establishes a framework for interrogation of enhancer function in living cells and supports an unexpected mechanism for enhancer control of Sox2 expression that uncouples transcription from enhancer proximity.
Highlights
Chromosomes are packaged and organized non-randomly within the mammalian nucleus
We find that the Sox2 promoter and SCR demonstrate similar spatial characteristics to nonregulatory regions in embryonic stem cells (ESCs), while differentiation of ESCs leads to significant compaction throughout the Sox2 region
This is a repressor system from the bacteria Pseudomonas putida that is involved in cumate metabolism and has been previously used as a tool for inducible gene expression (Mullick et al, 2006)
Summary
Chromosomes are packaged and organized non-randomly within the mammalian nucleus. Emerging evidence suggests that 3D genome topology plays a fundamental role in genome control, including the regulation of gene expression programs (Bickmore, 2013; Krijger and de Laat, 2016; Schwarzer and Spitz, 2014). Each chromosome occupies discrete chromosomal territories (Cremer et al, 2006). These territories are further structured into distinct compartments that separate active and repressive chromatin (LiebermanAiden et al, 2009; Sexton et al, 2012). Chromosomal contacts are disfavored across TAD boundaries. Most cell-type specific contacts occur within TAD boundaries, and disruption of TAD architecture leads to dysregulation of gene expression (Dowen et al, 2014; Gröschel et al, 2014; Guo et al, 2015; Lupiáñez et al., 2015; Narendra et al, 2015; Nora et al, 2017)
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