Scott Barolo

Shadow enhancers: Frequently asked questions about distributed cis‐regulatory information and enhancer redundancy

This paper, in the form of a frequently asked questions page (FAQ), addresses outstanding questions about “shadow enhancers”, quasi‐redundant cis‐regulatory elements, and their proposed roles in transcriptional control. Questions include: What exactly are shadow enhancers? How many genes have shadow/redundant/distributed enhancers? How redundant are these elements? What is the function of distributed enhancers? How modular are enhancers? Is it useful to study a single enhancer in isolation? In addition, a revised definition of “shadow enhancers” is proposed, and possible mechanisms of shadow enhancer function and evolution are discussed.

Structural Rules and Complex Regulatory Circuitry Constrain Expression of a Notch- and EGFR-Regulated Eye Enhancer

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? Here, we examine in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparklings regulatory sites converts it into a robust photoreceptor-specific enhancer. Our results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancers organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others.

The cis-Regulatory Logic of Hedgehog Gradient Responses: Key Roles for Gli Binding Affinity, Competition, and Cooperativity

Reporter gene assays in Drosophila and modeling reveal mechanisms that control transcriptional responses to gradients of Hedgehog ligands. Interpreting a Gradient Through Cooperative Repression Hedgehog (Hh) signaling is required for tissue patterning during development. In the absence of Hh, the transcription factor Cubitus interruptus (Ci) in Drosophila is cleaved to generate a repressor form, whereas it is converted to an activator form in the presence of Hh. Gradients of ligands of the Hh family thus produce opposing gradients of repressor and activator forms of Ci. Both forms of Ci bind to the same enhancer elements in the promoter regions of target genes. One model of Hh morphogen activity implies that high-affinity binding sites for Ci should confer a broader expression domain for a gene. However, Parker et al. (see also the Perspective by Whitington et al.) noted that the enhancer of a broadly expressed Hh target gene decapentaplegic (dpp) contains low-affinity sites for Ci, whereas high-affinity sites are present in the enhancer of a Hh target gene with a more restricted expression pattern, patched (ptc). Reporter gene assays indicated that low-affinity binding sites for Ci were required for dpp to be expressed in areas of low Hh signal in Drosophila imaginal discs. Replacing the low-affinity Ci binding sites in the dpp enhancer with the higher-affinity Ci binding sites from ptc limited expression of dpp to areas of high Hh signal and caused severe developmental defects. Computational modeling, supported by in vivo experiments in flies, suggested that these results were best explained by cooperative binding of the repressor forms of Ci to enhancers and predicted that a single high-affinity site would mediate gene transcription in response to intermediate Hh signal, whereas three high-affinity sites would cause transcriptional repression. Thus, transcriptional responses to gradients of Hh are shaped by competition between repressor and activator forms for binding sites, the affinity of transcription factor binding sites, and cooperative binding of repressor forms. Gradients of diffusible signaling proteins control precise spatial patterns of gene expression in the developing embryo. Here, we use quantitative expression measurements and thermodynamic modeling to uncover the cis-regulatory logic underlying spatially restricted gene expression in a Hedgehog (Hh) gradient in Drosophila. When Hh signaling is low, the Hh effector Gli, known as Cubitus interruptus (Ci) in Drosophila, acts as a transcriptional repressor; when Hh signaling is high, Gli acts as a transcriptional activator. Counterintuitively and in contrast to previous models of Gli-regulated gene expression, we found that low-affinity binding sites for Ci were required for proper spatial expression of the Hh target gene decapentaplegic (dpp) in regions of low Hh signal. Three low-affinity Ci sites enabled expression of dpp in response to low signal; increasing the affinity of these sites restricted dpp expression to regions of maximal signaling. A model incorporating cooperative repression by Ci correctly predicted the in vivo expression of a reporter gene controlled by a single Ci site. Our work clarifies how transcriptional activators and repressors, competing for common binding sites, can transmit positional information to the genome. It also provides an explanation for the widespread presence of conserved, nonconsensus Gli binding sites in Hh target genes.

Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients. We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp, wingless and stripe, by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.

A model of spatially restricted transcription in opposing gradients of activators and repressors

Morphogens control patterns of transcription in development, often by establishing concentration gradients of a single transcriptional activator. However, many morphogens, including Hedgehog, create opposing activator and repressor gradients (OARGs). In contrast to single activator gradients, it is not well understood how OARGs control transcriptional patterns. We present a general thermodynamic model that explains how spatial patterns of gene expression are established within OARGs. The model predicts that differences in enhancer binding site affinities for morphogen‐responsive transcription factors (TFs) produce discrete transcriptional boundaries, but only when either activators or repressors bind cooperatively. This model quantitatively predicts the boundaries of gene expression within OARGs. When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila. Because our model is general, it may explain the role of low‐affinity binding sites in many contexts, including mammalian Hedgehog gradients.

Regulation of the feedback antagonist naked cuticle by Wingless signaling

Signaling pathways usually activate transcriptional targets in a cell type-specific manner. Notable exceptions are pathway-specific feedback antagonists, which serve to restrict the range or duration of the signal. These factors are often activated by their respective pathways in a broad array of cell types. For example, the Wnt ligand Wingless (Wg) activates the naked cuticle (nkd) gene in all tissues examined throughout Drosophila development. How does the nkd gene respond in such an unrestricted manner to Wg signaling? Analysis in cell culture revealed regions of the nkd locus that contain Wg response elements (WREs) that are directly activated by the pathway via the transcription factor TCF. In flies, Wg signaling activates these WREs in multiple tissues, in distinct but overlapping patterns. These WREs are necessary and largely sufficient for nkd expression in late stage larval tissues, but only contribute to part of the embryonic expression pattern of nkd. These results demonstrate that nkd responsiveness to Wg signaling is achieved by several WREs which are broadly (but not universally) activated by the pathway. The existence of several WREs in the nkd locus may have been necessary to allow the Wg signaling-Nkd feedback circuit to remain intact as Wg expression diversified during animal evolution.

An ancient yet flexible cis-regulatory architecture allows localized Hedgehog tuning by patched/Ptch1

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure—one of the oldest cis-regulatory features discovered in animal genomes—explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways. DOI: http://dx.doi.org/10.7554/eLife.13550.001

Using the Game of Mastermind to Teach, Practice, and Discuss Scientific Reasoning Skills

The code-breaking game Mastermind, which can be played in minutes at no cost, creates opportunities for students to discuss scientific reasoning, hypothesis-testing, effective experimental design, and sound interpretation of results.

How to tune an enhancer.

Every cell’s genome contains two main classes of functional DNA. The best understood type of DNA sequence, which was also the first to be discovered, is that which encodes RNA and protein products via the near-universal “genetic code” (1). A more mysterious but equally important class of functional DNA is cis-regulatory sequence, which does not have a physical product but, instead, encodes the conditions under which a particular RNA will be produced. Cis-regulatory DNA sequences are the primary (although not the only) determinant of gene expression: Not only the rate of RNA production but also the timing, spatial patterning, and environmental control of every gene’s activity are largely controlled by these DNA sequences, which are usually in the general vicinity of the gene they regulate. These DNA segments (often called enhancers) have no enzymatic activity on their own, but act as scaffolds for large complexes of proteins and RNAs that directly control the activity of a gene’s promoter, sometimes over distances of 1 million base pairs or more (2). Transcription factors (TFs), key protein regulators of gene expression, bind DNA in a sequence-specific manner, which means that the nature of the complex assembled at a given enhancer at a given time depends on its DNA sequence (cis information), in conjunction with the set of TFs present and active in the cell …