Live-cell imaging reveals enhancer-dependent Sox2 transcription in the absence of enhancer proximity

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.

How the Genome Folds: The Biophysics of Four-Dimensional Chromatin Organization

“…On the other hand, both cohesin and CTCF depletion only show minor effects on gene expression over a 6 hour window, suggesting that on a population scale, transcription is already potentiated and proceeds as before, even in the absence of TAD delimitation (83,97). These findings are corroborated by recent single locus imaging in mouse embryonic stem cells, in which no correlation was found between enhancer-promoter distance and transcript production (3). It also remains unclear to what extent specific histone modifications could mediate this potentiation.…”

BET inhibition disrupts transcription but retains enhancer-promoter contact

“…Maintenance of enhancer-promoter looping is thought to be crucial for gene activation, but emerging evidence and the data presented here suggest that enhancer-promoter contact and gene activation may be partially separable events. Importantly, physical interaction with the promoter may not be necessary for all enhancers 5,6 , although it appears to be a requisite for most. At the same time, enhancer-promoter looping alone is not sufficient for activation, as enhancer-promoter contacts have been observed in the absence of transcription 57,58 .…”

“…Enhancers can be situated far away from the genes they regulate 1,4 . Although not always the case 5,6 , at many gene loci proximity between enhancers and promoters is thought to be essential for enhancer function and gene activation 7,8 . How these enhancer-promoter interactions are initiated and maintained is not clearly understood.…”

Decoding the Notch signal

“…Some studies have correlated bursting frequency with enhancer-promoter interactions via induced looping (Bartman, Hsu, Hsiung, Raj, & Blobel, 2016). Others find no correlation between enhancer-promoter proximity and transcriptional activity (Alexander et al, 2019;Chen et al, 2018), suggesting other mechanisms are involved. However, the outcome, in terms of total number of mRNA molecules produced in a single bursting event (bursting size), has been correlated with the lifetime of PolII clusters ( Figure 2b; Cho et al, 2016), suggesting that the formation of these clusters is an important step and hence a likely consequence from NICD recruitment.…”