Shadow enhancers enable Hunchback bifunctionality in the Drosophila embryo

Abstract

Hunchback (Hb) is a bifunctional transcription factor that activates and represses distinct enhancers. Here, we investigate the hypothesis that Hb can activate and repress the same enhancer. Computational models predicted that Hb bifunctionally regulates the even-skipped (eve) stripe 3+7 enhancer (eve3+7) in Drosophila blastoderm embryos. We measured and modeled eve expression at cellular resolution under multiple genetic perturbations and found that the eve3+7 enhancer could not explain endogenous eve stripe 7 behavior. Instead, we found that eve stripe 7 is controlled by two enhancers: the canonical eve3+7 and a sequence encompassing the minimal eve stripe 2 enhancer (eve2+7). Hb bifunctionally regulates eve stripe 7, but it executes these two activities on different pieces of regulatory DNA-it activates the eve2+7 enhancer and represses the eve3+7 enhancer. These two "shadow enhancers" use different regulatory logic to create the same pattern.enhancer | computational model | bifunctional transcription factor | Drosophila development | Hunchback T ranscription factors (TFs) are typically categorized as activators or repressors, but many TFs can act bifunctionally by both activating and repressing target genes (1-4). Changes in TF activity can result from posttranslational modifications, protein cleavage, or translocation of cofactors into the nucleus (5-7). However, in cases where a TF activates and represses genes in the same cells, bifunctionality is controlled by enhancer sequences, which are responsible for tissue-specific gene expression (8). For example, in Drosophila, Dorsal activates genes when it binds to enhancers alone or near Twist (9, 10) but represses genes when it binds near other TFs (11-13). The DNA sequence of a TF's binding site can also alter TF activity [e.g., the glucocorticoid receptor (14, 15)]. Identifying how the activity of bifunctional TFs is controlled will be critical for inferring accurate gene regulatory networks from genomic data (16).Here, we investigate how TF bifunctionality is controlled using a classic example: the Drosophila gene hunchback (hb) (1, 17, 18). Hb both activates and represses even-skipped (eve) by acting on multiple enhancers. Hb activates eve stripes 1 and 2 and represses stripes 4, 5, and 6 (19-22). Computational models from us and others support the hypothesis that Hb both activates and represses the enhancer that controls eve stripes 3 and 7 (eve3+7) (Fig. 1) (22-25).In contrast to others, our computational models of eve3+7 activity do not include regulatory DNA sequence (26-30). Instead, our modeling approach uses regression to identify the activators and repressors that control a given pattern; we refer to the identity and role of the regulators as "regulatory logic." Modeling regulatory logic without including DNA sequence enables a powerful strategy to dissect gene regulation in a complex locus. We can compare the regulatory logic of an enhancer reporter pattern to that of the corresponding portion of the endogenous pattern to determine whether the an...

Krüppel Expression Levels Are Maintained through Compensatory Evolution of Shadow Enhancers

“…However, even if these interactions are not direct, the different responses of the proximal and distal enhancers to perturbation show that these enhancers use different regulatory logic, contradicting the initial picture of shadow enhancers as binding to the same set of TFs (Hong et al, 2008; Barolo, 2012). Our emerging picture is that shadow enhancers each build the same pattern in different ways; previous studies have shown that the snail shadow enhancers respond differently to a single repressor, (Dunipace et al, 2011), and in the eve locus, hb activates one shadow enhancer for stripe 7 and represses the other (Staller et al, 2015b). Here, we find an even more dramatic difference in the regulatory logic of the Kr shadow enhancers: each is activated by a non-overlapping set of TFs.…”

Combinatorial Gene Regulation through Kinetic Control of the Transcription Cycle

“…These “shadow” enhancers individually generate similar patterns of expression, but their combined expression levels are unpredictable (Bothma et al 2015; Dunipace et al 2011). We have shown previously that individual shadow enhancers can respond to different activators (Staller et al 2015; Wunderlich et al 2015). This suggests that shadow enhancers might work on different steps in transcription, in which case the kinetic model that we propose here could help to predict their combined activity.…”

Stepwise Progression of Embryonic Patterning

“…Recent studies have found that multiple cis-regulatory modules are often associated with genes and are co-acting [78–81]. Some CRMs work concurrently to control spatial domains and levels of expression [82, 83], whereas others work sequentially to control the changing expression of genes in time [84].…”