Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

What is an enhancer?

“…Therefore, the identification of putative enhancer elements based on epigenomic mapping allows scientists, specifically developmental biologists, to reconstruct the networks underlying gene expression changes during developmental transitions and in cell populations that were previously not tractable. However, not all putative enhancers identified based on their chromatin signature indeed affect gene expression at their endogenous locus [35][36][37][38] or are capable of activating a reporter gene in episomal enhancer assays [38][39][40] or when integrated randomly into the genome, 38 questioning their status as an enhancer.…”

“…54 Enhancer assays rely on a cut-off to distinguish elements without intrinsic activity from elements with intrinsic activity, and in some cases have limited sensitivity and/or high variability between replicates, specifically when the whole genome is assayed. This can make it difficult to reliably characterize weak enhancers that might play an important role when combined with other weak enhancers at their endogenous locus [36][37][38] : many identified elements with typical enhancer chromatin signatures are not sufficient to activate gene expression but can do so when combined with other enhancers [36][37][38] (see below for a more detailed description). Together, this cautions against over-interpreting negative outcomes of MPRAs: there are many reasons why an individual sequence does not activate reporter gene expression in a given assay; this does not necessarily mean that the assayed DNA element cannot contribute to target gene expression in a different context.…”

“…35,36 In other cases, certain elements can boost target gene expression in the presence of other enhancers but fail to do so on their own. 12,36,37 Based on reduced target gene expression in their absence, these elements might be classified as enhancers if no additional experiments were performed. However, as these elements are not sufficient to activate target gene expression, they do not fall under the original enhancer definition and it is up for debate whether these elements should be referred to as enhancers, or whether these "enhancer-like" elements represent a class of their own that activates transcription via distinct mechanisms.…”

“…Some of these clusters are referred to as super-enhancers, 62 stretch enhancers, 63 or locus control regions 64 depending on their levels of co-activator binding or for historical reasons. Their constituent elements can work together in a redundant, 36 additive, 37,55 or super-additive [36][37][38]57,65 fashion. Interestingly, many individual elements of enhancer clusters qualify as "enhancer-like" elements, as they either fail to activate their target gene in the absence of additional enhancers at the endogenous locus and/or have no activity in plasmid-based reporter assays 12,[36][37][38]56 Despite low intrinsic enhancer activity, deletion of these elements at their endogenous locus can lead to strongly reduced expression of the target genes.…”

“…Their constituent elements can work together in a redundant, 36 additive, 37,55 or super-additive [36][37][38]57,65 fashion. Interestingly, many individual elements of enhancer clusters qualify as "enhancer-like" elements, as they either fail to activate their target gene in the absence of additional enhancers at the endogenous locus and/or have no activity in plasmid-based reporter assays 12,[36][37][38]56 Despite low intrinsic enhancer activity, deletion of these elements at their endogenous locus can lead to strongly reduced expression of the target genes. The molecular mechanisms underlying this behavior remain unclear and might vary from locus to locus.…”