Importantly, enhancer activity is highly specific across cell types ( Heintzman et al., 2009 ENCODE Project Consortium, 2012 Zhu et al., 2013) and modulated during cellular differentiation ( Blum et al., 2012 Buecker et al., 2014 Huang et al., 2016 Wamstad et al., 2012), and this activity correlates with nearby gene expression. These features facilitate transcription factor occupancy, enrichment of co-activators such as p300 and Mediator, and transcription of non-coding enhancer RNAs (eRNAs), all of which play important roles in modulation of target gene expression ( Kim et al., 2015 Long et al., 2016). Enhancers demonstrate unique epigenetic markings, enriched for H3K4me1 and H3K27ac ( Creyghton et al., 2010 Heintzman et al., 2007 Rada-Iglesias et al., 2011), and are highly accessible, as demonstrated by elevated DNase sensitivity and transposition susceptibility ( Boyle et al., 2008 Buenrostro et al., 2013 Thurman et al., 2012).
First discovered within a metazoan genome over three decades ago ( Banerji et al., 1983), it is now predicted that greater than 300,000 enhancers are encoded in the human genome ( ENCODE Project Consortium, 2012 Zhu et al., 2013). Within this 3D framework, gene expression programs are established by non-coding regulatory enhancer elements. Thus, 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). Chromosomal contacts are disfavored across TAD boundaries. At finer scales, chromosomes are partitioned into largely-invariant, sub-megabase sized topologically-associated domains (TADs), which break up the linear genome into interactive neighborhoods ( Dixon et al., 2012 Nora et al., 2012).
These territories are further structured into distinct compartments that separate active and repressive chromatin ( Lieberman-Aiden et al., 2009 Sexton et al., 2012). Within the nucleus, each chromosome occupies discrete chromosomal territories ( Cremer et al., 2006). 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). 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.Ĭhromosomes are packaged and organized non-randomly within the mammalian nucleus. 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. 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. 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). However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Enhancers are important regulatory elements that can control gene activity across vast genetic distances.
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