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...

A sequence level model of an intact locus predicts the location and function of nonadditive enhancers

“…This data corresponded well to RNA data collected in 3D from CantonS [28]. Quanititative enhancer-reporter data was obtained from Staller et al [29]. Relative transcription levels along the AP axis were obtained from the FlyEx database [30–33].…”

“…For the remainder of the work we used the sequences reported in Staller et al [29] as these sequences can be directly compared quantitative to enhancer-reporter data from the same work.…”

“…The eve RNA was imaged together with Eve protein so that the protein channel could be used for data registration. In order to use data from Staller et al [29] nuclei in a 10% DV strip along AP axis were registered to the FlyEx database using the eve RNA channel. All data was registered using the BREReA [38, 39] software.…”

Molecular aspects of zygotic embryogenesis in sunflower (Helianthus annuus L.): correlation of positive histone marks with HaWUS expression and putative link HaWUS/HaL1L

“…Bifunctional TFs, depending on the target gene, are known both in animals (Adkins et al 2006;Staller et al 2015) and plants. For example, Pti4, a transcriptional activator of tomato (Solanum lycopersicum), and Arabidopsis WRKY53, activate or repress the expression of genes, depending on the nature of the promoter sequence of their target genes (Miao et al 2004;González-Lamothe et al 2008).…”

Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies

“…the ability for individuals in a population to produce similar phenotypes regardless of environmental or genetic perturbation) (Waddington, 1942). Buffering of gene expression occurs at the levels of cis-regulatory logic (Dunipace et al, 2013;Frankel et al, 2010;Hong et al, 2008;Staller et al, 2015;Wunderlich et al, 2015) and gene networks (Cassidy et al, 2013;Lott et al, 2007;Manu et al, 2009). Dark shadow enhancers are an interesting integration of these mechanisms, whereby a primary enhancer induces expression of a factor that feeds back to repress a dark shadow enhancer.…”