Author response: Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo

Abstract

Metazoan genes are embedded in a rich milieu of regulatory information that often includes multiple enhancers possessing overlapping activities. In this study, we employ quantitative live imaging methods to assess the function of pairs of primary and shadow enhancers in the regulation of key patterning genes-knirps, hunchback, and snail-in developing Drosophila embryos. The knirps enhancers exhibit additive, sometimes even super-additive activities, consistent with classical gene fusion studies. In contrast, the hunchback enhancers function sub-additively in anterior regions containing saturating levels of the Bicoid activator, but function additively in regions where there are diminishing levels of the Bicoid gradient. Strikingly sub-additive behavior is also observed for snail, whereby removal of the proximal enhancer causes a significant increase in gene expression. Quantitative modeling of enhancer-promoter interactions suggests that weakly active enhancers function additively while strong enhancers behave sub-additively due to competition with the target promoter.

Dynamic patterning by morphogens illuminated by cis-regulatory studies

“…In addition, enhancer interactions not only help to refine spatial patterns, but can also function to modulate levels of gene expression. For example, some genes, such as kni, sna and Kr, require balanced input from two enhancers (Bothma et al, 2015;Scholes et al, 2019). In the case of kni, two enhancers act in an The proximal (yellow) and distal (gold) enhancers that are bound by Bcd to drive hb gene expression ( pink) are active in similar domains, but the distal enhancer expression, specifically, has a repressive input that acts in a dominant fashion (Perry et al, 2012).…”

“…In the case of kni, two enhancers act in an The proximal (yellow) and distal (gold) enhancers that are bound by Bcd to drive hb gene expression ( pink) are active in similar domains, but the distal enhancer expression, specifically, has a repressive input that acts in a dominant fashion (Perry et al, 2012). (B) kni levels (teal) are supported by both proximal and distal enhancers, and each enhancer equally contributes to support kni levels (Bothma et al, 2015). (C) Proximal and distal enhancers at the sna locus act together to regulate levels of expression (brown) in a non-additive (or sub-additive) manner (Bothma et al, 2015;Dunipace et al, 2011).…”

Dynamic regulation of anterior-posterior patterning genes in livingDrosophilaembryos

“…Consistent with previous results [35,36], enhancers appear to be responsible for successive induction of transcriptional bursting because removal of hb shadow enhancer specifically altered the length of transcription period without changing the transcription rate ( Figure 3C and D). Previous transgene assay suggested that hb shadow enhancer is ~2-fold weaker than proximal enhancer [30]. Genome-editing analysis in this study revealed that the deletion of shadow enhancer causes ~2-fold increase in the level of total RNA production ( Figure 3B), suggesting that interaction between P2 promoter and strong proximal enhancer is competitively blocked by distal weak enhancer to downregulate de novo RNA synthesis and facilitate refinement of initial expression domain during nc14.…”

“…Gap genes are typically regulated by two redundant enhancers that can produce overlapping patterns in early embryos [27]. While previous transgene assay suggested that redundant enhancers foster robustness of gene expression [28,29] or help to increase transcriptional output [30], it remains unclear if they can simultaneously interact with target promoter. Anterior expression of hb is controlled not only by classical proximal enhancer [24,31], but also by newly-identified distal redundant enhancer, or shadow enhancer [27] ( Figure 3A).…”

“…Antagonistic relationship of hb enhancers suggests that they do not form common "transcription hubs" for gene activation, which differs from cooperative action of super enhancers in mammalian cells [37][38][39]. Regulation of hb is also unique from enhancer competition mechanism seen at other locus [30,40] in a point that distal weak enhancer sequesters interaction with target promoter from remote location despite its "topological disadvantage" over strong proximal enhancer. It might be possible that transcriptional interference by hb shadow enhancer helps subsequent recruitment of distal stripe enhancer to the promoter region at late nc14 [41].…”