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      Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity

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          Developmental enhancers integrate graded concentrations of transcription factors (TFs) to create sharp gene expression boundaries. Here we examine the hunchback P2 (HbP2) enhancer which drives a sharp expression pattern in the Drosophila blastoderm embryo in response to the transcriptional activator Bicoid (Bcd). We systematically interrogate cis and trans factors that influence the shape and position of expression driven by HbP2, and find that the prevailing model, based on pairwise cooperative binding of Bcd to HbP2 is not adequate. We demonstrate that other proteins, such as pioneer factors, Mediator and histone modifiers influence the shape and position of the HbP2 expression pattern. Comparing our results to theory reveals how higher-order cooperativity and energy expenditure impact boundary location and sharpness. Our results emphasize that the bacterial view of transcription regulation, where pairwise interactions between regulatory proteins dominate, must be reexamined in animals, where multiple molecular mechanisms collaborate to shape the gene regulatory function.

          eLife digest

          Building an organism from scratch requires genes to be switched on or off at precisely the right time, in the right place, and at the right level. Enhancers are stretches of DNA that work as switches to turn on target genes. For instance, in the front part of fruit fly embryos, the P2 enhancer switches on a gene called Hunchback, which is crucial for development.

          A number of molecular actors, including proteins called transcription factors, work together to turn on genes by interacting with enhancers. Genes like Hunchback can turn on suddenly, even though they are controlled by transcription factors whose levels are changing gradually: in other words, if Hunchback were controlled by a light switch with a dimmer, the light would suddenly come on as the dimmer was gradually moved up. For enhancers, the question is how transcription factors interact with DNA to convert a gradual input into an abrupt, sharp switch. A commonly accepted view is that Hunchback is turned on when molecules of a transcription factor called Bicoid help each other to bind to multiple binding sites on the P2 enhancer.

          Park et al. investigated this mechanism by examining how the Hunchback gene responded to changes in the sequence of the P2 enhancer, and to changes in the levels of regulatory proteins that bind to it. The resulting observations were then compared to mathematical models that simulate turning on Hunchback under different conditions. The experiments revealed that, in fact, switching on Hunchback requires more than Bicoid proteins helping each other to bind on the P2 enhancers. Molecules other than Bicoid were also needed, and the cell also potentially had to burn energy.

          Variations in the sequence of enhancers are linked to evolution of new species but also to problems in development or even diseases such as cancer. Understanding precisely how these sequences turn on genes will give us insight into which types of changes are important for disease and evolution.

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          Most cited references 90

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          Transcription factors: from enhancer binding to developmental control.

          Developmental progression is driven by specific spatiotemporal domains of gene expression, which give rise to stereotypically patterned embryos even in the presence of environmental and genetic variation. Views of how transcription factors regulate gene expression are changing owing to recent genome-wide studies of transcription factor binding and RNA expression. Such studies reveal patterns that, at first glance, seem to contrast with the robustness of the developmental processes they encode. Here, we review our current knowledge of transcription factor function from genomic and genetic studies and discuss how different strategies, including extensive cooperative regulation (both direct and indirect), progressive priming of regulatory elements, and the integration of activities from multiple enhancers, confer specificity and robustness to transcriptional regulation during development.
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            Mutations affecting segment number and polarity in Drosophila

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              Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

              The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

                Author and article information

                Role: Reviewing Editor
                Role: Senior Editor
                eLife Sciences Publications, Ltd
                21 June 2019
                : 8
                [1 ]deptDepartment of Systems Biology Harvard Medical School BostonUnited States
                Michigan State University United States
                University of Michigan United States
                Michigan State University United States
                Author notes

                Novartis Institutes for Biomedical Research, Cambridge, United States.


                Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States.


                Department of Developmental and Cell Biology, University of California Irvine, Irvine, United States.

                © 2019, Park et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Funded by: FundRef, National Institutes of Health;
                Award ID: 5K99HD073191-02
                Award Recipient :
                Funded by: FundRef, National Institutes of Health;
                Award ID: R01GM122928
                Award Recipient :
                Funded by: FundRef, Giovanni Armenise-Harvard Foundation;
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Research Article
                Chromosomes and Gene Expression
                Computational and Systems Biology
                Custom metadata
                The sharp expression pattern driven by a classic, simple animal enhancer is determined by multiple molecular mechanisms, not only cooperative binding of the activating transcription factor as was previously thought.

                Life sciences

                d. melanogaster, drosophila, enhancer, transcription


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