Abstract
Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based 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.
Highlights
Gene expression is regulated by two types of genetic elements: Trans elements typically encode proteins such as transcription factors (TFs), which subsequently bind cis-regulatory elements (CREs) that must be on the same DNA molecule as the gene they regulate
We refer to this element as a CRE rather than an SE because one group has previously reported that this element is an alternative promoter [17]
To identify if this element is required for Nanog expression, we used genomic editing to insert a tamoxifen (4OHT)-inducible Cre-recombinase (CreERT2) into the constitutively expressed Rosa26 locus in embryonic stem cells (ESC) to facilitate conditional deletions and biallelically inserted loxP sites to flank a 2.5-kb region of the −5 CRE to encompass two Nanog, Oct4, and Sox2 (NOS) binding sites (Fig. S1A, Fig 1A, left)
Summary
The −5 Nanog CRE is required for embryonic stem cell pluripotency in a Nanog-dependent manner. In at least one report the −5 CRE was considered an alternative promoter that played a critical role in regulating pluripotency through an alternative Nanog isoform [17] Given this ambiguity we chose to definitively establish if this element had enhancer activity within the context of normal chromatin with our 4OHT-inducible Cre-LoxP system by inserting one of the LoxP sites in the opposite orientation (Fig. 3A, left). Deletion of either the 5’ or 3’ constituent enhancer results in an approximately 50% reduction in Nanog mRNA, but these reductions were insufficient to alter pluripotency as measured by Oct, Esrrb, or Klf expression (Fig. 4C) This demonstrates that neither the 5’ nor the 3’ constituent enhancer is required for pluripotency, even though together they promote normal Nanog expression. We performed single-cell RTqPCR on the −5 SE biallelically deleted cells with Nanog in
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