Defining a critical enhancer near Nanog using chromatin-focused approaches identifies RNA Pol II recruitment as required for expression

Abstract

Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based reporter assays of gene expression. Currently, histone marks are used heavily to identify and define enhancers but both methods 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 (CRE) which has been reported by others to be either an alternative promoter or a super-enhancer (SE). We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We also demonstrate that the dominant SE 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. AUTHOR SUMMARYDifferent DNA elements help regulate the levels of gene expression. One such element are enhancers, short sequences that interact with genes to modulate levels of expression but can operate over large distances. Previously, these sequences were defined by their ability to regulate expression independent of their distance from a gene and the orientation of the sequence. However, these characteristics were found using techniques that did not recapitulate the native environment. Here, we have shown that an enhancer of one gene is indeed an enhancer by testing its distance and orientation-independence within the native environment. We also show that the mechanisms by which the enhancer is regulating expression is by controlling the levels of RNA Polymerase II at a gene. RNA Polymerase II is the protein that converts the gene sequence to a form usable by a cell, called mRNA. This is interesting because while this has been considered historically the main way enhancers operate, more recent work has focused on other, later regulatory steps involved in controlling mRNA production.