Genome editing demonstrates that the −5 kb Nanog enhancer regulates Nanog expression by modulating RNAPII initiation and/or recruitment

Abstract

Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmidbased reporter assays of gene expression. At present, histone marks are used to identify and define enhancers but do not consider the endogenous role of an enhancer in the context of native chromatin. We employed a combination of genomic editing, single cell analyses, and sequencing approaches to investigate a Nanog-associated cis-regulatory element, which has been reported by others to be either an alternative promoter or a super-enhancer. We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We next demonstrate that the dominant super-enhancer modulates Nanog globally and operates by recruiting and/or initiating RNA Polymerase II. Our studies have important implications to how transcriptional enhancers are defined and how they regulate gene expression. This article contains supporting information. ‡ Equal contribution.

Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome

“…Some authors consider enhancers combinations of multiple DHSs or longer stretches of DNA sequences. However, other studies have shown that the activity of these long enhancers can be reproduced by shorter versions of ~500 bp [40,41]. Coordinates of all tested genomic fragments are provided in Supplementary Dataset 1.…”

Super-Enhancers and CTCF in Early Embryonic Cell Fate Decisions

“…The master pluripotency TF Nanog is near three SEs (−5SE, −45SE, +60SE, named based on distance from Nanog promoter in kb; Figure 1A; Pulakanti et al, 2013;Whyte et al, 2013;Blinka et al, 2016;Agrawal et al, 2021). These SEs and the numerous CTCF sites at the locus make it an ideal model system to understand how CREs regulate cell fate decisions by modulating Nanog expression (Blinka and Rao, 2017).…”

“…Circular Chromosome Capture (3C) of the three SEs at the Nanog locus demonstrates that they physically interact with the Nanog promoter, but CRISPR-based deletion of each SE differentially alters expression (Blinka et al, 2016). Deletion of the −45SE causes an approximately 50% decrease in Nanog expression, deletion of the −5SE causes a nearly 90% decrease, but deletion of the +60SE causes no change (Figure 1B; Blinka et al, 2016;Agrawal et al, 2021). This leads to the important question: through what mechanism(s) do the −5SE and −45SE regulate Nanog, expression?…”

“…These sub-TADs are found to contain tissue or developmentspecific genes. Insulated neighborhoods (INs) are among the (Blinka et al, 2016;Agrawal et al, 2021). (C) The interaction between the −5SE and Nanog and how it alters RNAPII dynamics (Agrawal et al, 2021).…”

“…Schematic of a theoretical loop containing all three SEs and Nanog depicting. (B) The changes in Nanog expression upon deletion(Blinka et al, 2016;Agrawal et al, 2021). (C) The interaction between the −5SE and Nanog and how it alters RNAPII dynamics(Agrawal et al, 2021).…”