Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.
ABSTRACT: Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. We investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, we found that in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. We also found evidence that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. Our results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. We discuss how counter-repression may influence eve stripe 2 regulation and evolution.
Project description: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.
Project description:Changes in regulatory DNA contribute to phenotypic differences within and between taxa. Comparative studies show that many transcription factor binding sites (TFBS) are conserved between species whereas functional studies reveal that some mutations segregating within species alter TFBS function. Consistently, in this analysis of 13 regulatory elements in Drosophila melanogaster populations, single base and insertion/deletion polymorphism are rare in characterized regulatory elements. Experimentally defined TFBS are nearly devoid of segregating mutations and, as has been shown before, are quite conserved. For instance 8 of 11 Hunchback binding sites in the stripe 3+7 enhancer of even-skipped are conserved between D. melanogaster and Drosophila virilis. Oddly, we found a 72 bp deletion that removes one of these binding sites (Hb8), segregating within D. melanogaster. Furthermore, a 45 bp deletion polymorphism in the spacer between the stripe 3+7 and stripe 2 enhancers, removes another predicted Hunchback site. These two deletions are separated by ?250 bp, sit on distinct haplotypes, and segregate at appreciable frequency. The Hb8? is at 5 to 35% frequency in the new world, but also shows cosmopolitan distribution. There is depletion of sequence variation on the Hb8?-carrying haplotype. Quantitative genetic tests indicate that Hb8? affects developmental time, but not viability of offspring. The Eve expression pattern differs between inbred lines, but the stripe 3 and 7 boundaries seem unaffected by Hb8?. The data reveal segregating variation in regulatory elements, which may reflect evolutionary turnover of characterized TFBS due to drift or co-evolution.
Project description:Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected "stripe" enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. Here, we employ BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. We propose that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient.
Project description:The regulation of segmentation gene expression is investigated by computational modeling using quantitative expression data. Previous tissue culture assays and transgene analyses raised the possibility that Hunchback (Hb) might function as both an activator and repressor of transcription. At low concentrations, Hb activates gene expression, whereas at high concentrations it mediates repression. Under the same experimental conditions, transcription factors encoded by other gap genes appear to function as dedicated repressors. Models based on dual regulation suggest that the Hb gradient can be sufficient for establishing the initial Kruppel (Kr) expression pattern in central regions of the precellular embryo. The subsequent refinement of the Kr pattern depends on the combination of Hb and the Giant (Gt) repressor. The dual-regulation models developed for Kr also explain some of the properties of the even-skipped (eve) stripe 3+7 enhancer. Computational simulations suggest that repression results from the dimerization of Hb monomers on the DNA template.
Project description:Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. Here, we investigate the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. We also present evidence that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. We conclude that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo.
Project description:Comparative studies of developmental processes are one of the main approaches to evolutionary developmental biology (evo-devo). Over recent years, there has been a shift of focus from the comparative study of particular regulatory genes to the level of whole gene networks. Reverse-engineering methods can be used to computationally reconstitute and analyze the function and dynamics of such networks. These methods require quantitative spatio-temporal expression data for model fitting. Obtaining such data in non-model organisms remains a major technical challenge, impeding the wider application of data-driven mathematical modeling to evo-devo.We have raised antibodies against four segmentation gene products in the moth midge Clogmia albipunctata, a non-drosophilid dipteran species. We have used these antibodies to create a quantitative atlas of protein expression patterns for the gap gene hunchback (hb), and the pair-rule gene even-skipped (eve). Our data reveal differences in the dynamics of Hb boundary positioning and Eve stripe formation between C. albipunctata and Drosophila melanogaster. Despite these differences, the overall relative spatial arrangement of Hb and Eve domains is remarkably conserved between these two distantly related dipteran species.We provide a proof of principle that it is possible to acquire quantitative gene expression data at high accuracy and spatio-temporal resolution in non-model organisms. Our quantitative data extend earlier qualitative studies of segmentation gene expression in C. albipunctata, and provide a starting point for comparative reverse-engineering studies of the evolutionary and developmental dynamics of the segmentation gene system.
Project description:We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.
Project description:Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo.
Project description:Anterior-posterior (AP) body segmentation of the fruit fly (Drosophila) is first seen in the 7-stripe spatial expression patterns of the pair-rule genes, which regulate downstream genes determining specific segment identities. Regulation of pair-rule expression has been extensively studied for the even-skipped (eve) gene. Recent live imaging, of a reporter for the 2nd eve stripe, has demonstrated the stochastic nature of this process, with 'bursts' in the number of RNA transcripts being made over time. We developed a stochastic model of the spatial and temporal expression of eve stripe 2 (binding by transcriptional activators (Bicoid and Hunchback proteins) and repressors (Giant and Krüppel proteins), transcriptional initiation and termination; with all rate parameters constrained by features of the experimental data) in order to analyze the noisy experimental time series and test hypotheses for how eve transcription is regulated. These include whether eve transcription is simply OFF or ON, with a single ON rate, or whether it proceeds by a more complex mechanism, with multiple ON rates. We find that both mechanisms can produce long (multi-minute) RNA bursts, but that the short-time (minute-to-minute) statistics of the data is indicative of eve being transcribed with at least two distinct ON rates, consistent with data on the joint activation of eve by Bicoid and Hunchback. We also predict distinct statistical signatures for cases in which eve is repressed (e.g. along the edges of the stripe) vs. cases in which activation is reduced (e.g. by mutagenesis of transcription factor binding sites). Fundamental developmental processes such as gene transcription are intrinsically noisy; our approach presents a new way to quantify and analyze time series data during developmental patterning in order to understand regulatory mechanisms and how they propagate noise and impact embryonic robustness.
Project description:Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is "replaced" by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.