Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression.

Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves.

A novel role for trithorax in the gene regulatory network for a rapidly evolving fruit fly pigmentation trait.

Animal traits develop through the expression and action of numerous regulatory and realizator genes that comprise a gene regulatory network (GRN). For each GRN, its underlying patterns of gene expression are controlled by cis-regulatory elements (CREs) that bind activating and repressing transcription factors. These interactions drive cell-type and developmental stage-specific transcriptional activation or repression. Most GRNs remain incompletely mapped, and a major barrier to this daunting task is CRE identification. Here, we used an in silico method to identify predicted CREs (pCREs) that comprise the GRN which governs sex-specific pigmentation of Drosophila melanogaster. Through in vivo assays, we demonstrate that many pCREs activate expression in the correct cell-type and developmental stage. We employed genome editing to demonstrate that two CREs control the pupal abdomen expression of trithorax, whose function is required for the dimorphic phenotype. Surprisingly, trithorax had no detectable effect on this GRN's key trans-regulators, but shapes the sex-specific expression of two realizator genes. Comparison of sequences orthologous to these CREs supports an evolutionary scenario where these trithorax CREs predated the origin of the dimorphic trait. Collectively, this study demonstrates how in silico approaches can shed novel insights on the GRN basis for a trait's development and evolution.

Reporter gene assays and chromatin-level assays define substantially non-overlapping sets of enhancer sequences.

BACKGROUND: Transcriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence. RESULTS: We examine here whether these two types of definitions, activity-based and chromatin-based, effectively identify the same sets of sequences. We find that, concerningly, the overlap between the two groups is strikingly limited. Few of the data sets we compared displayed statistically significant overlap, and even for those, the degree of overlap was typically small (below 40% of sequences). Moreover, a substantial batch effect was observed in which experiment set rather than experimental method was a primary driver of whether or not chromatin-defined enhancers showed a strong overlap with reporter gene-defined enhancers. CONCLUSIONS: Our results raise important questions as to the appropriateness of both old and new enhancer definitions, and suggest that new approaches are required to reconcile the poor agreement among existing methods for defining enhancers.

Silencers, Enhancers, and the Multifunctional Regulatory Genome.

Negative regulation of gene expression by transcriptional silencers has been difficult to study due to limited defined examples. A new study by Gisselbrecht et al. has dramatically increased the number of identified silencers and reveals that they are bifunctional regulatory sequences that also act as gene expression-promoting enhancers.

Behavior-related gene regulatory networks: A new level of organization in the brain.

Neuronal networks are the standard heuristic model today for describing brain activity associated with animal behavior. Recent studies have revealed an extensive role for a completely distinct layer of networked activities in the brain-the gene regulatory network (GRN)-that orchestrates expression levels of hundreds to thousands of genes in a behavior-related manner. We examine emerging insights into the relationships between these two types of networks and discuss their interplay in spatial as well as temporal dimensions, across multiple scales of organization. We discuss properties expected of behavior-related GRNs by drawing inspiration from the rich literature on GRNs related to animal development, comparing and contrasting these two broad classes of GRNs as they relate to their respective phenotypic manifestations. Developmental GRNs also represent a third layer of network biology, playing out over a third timescale, which is believed to play a crucial mediatory role between neuronal networks and behavioral GRNs. We end with a special emphasis on social behavior, discuss whether unique GRN organization and cis-regulatory architecture underlies this special class of behavior, and review literature that suggests an affirmative answer.

Studying Transcriptional Enhancers: The Founder Fallacy, Validation Creep, and Other Biases.

Transcriptional enhancers play a major role in regulating metazoan gene expression. Recent developments in genomics and next-generation sequencing have accelerated and revitalized the study of this important class of sequence elements. Increased interest and attention, however, has also led to troubling trends in the enhancer literature. In this Opinion, I describe some of these issues and show how they arise from shifting and nonuniform enhancer definitions, and genome-era biases. I discuss how they can lead to interpretative errors and an unduly narrow focus on certain aspects of enhancer biology to the potential exclusion of others.

Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification.

Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). We demonstrate here that for the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. Our results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that, accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lame duck and is likely a member of the ETS transcription factor family. Our findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a crucial mechanism for determining unique responses to Ras signaling.

Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum.

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.

REDfly: the transcriptional regulatory element database for Drosophila.

The REDfly database provides a comprehensive curation of experimentally-validated Drosophila transcriptional cis-regulatory elements and includes information on DNA sequence, experimental evidence, patterns of regulated gene expression, and more. Now in its thirteenth year, REDfly has grown to over 23 000 records of tested reporter gene constructs and 2200 tested transcription factor binding sites. Recent developments include the start of curation of predicted cis-regulatory modules in addition to experimentally-verified ones, improved search and filtering, and increased interaction with the authors of curated papers. An expanded data model that will capture information on temporal aspects of gene regulation, regulation in response to environmental and other non-developmental cues, sexually dimorphic gene regulation, and non-endogenous (ectopic) aspects of reporter gene expression is under development and expected to be in place within the coming year. REDfly is freely accessible at http://redfly.ccr.buffalo.edu, and news about database updates and new features can be followed on Twitter at @REDfly_database.

PeakMatcher facilitates updated Aedes aegypti embryonic cis-regulatory element map.

BACKGROUND: The Aedes aegypti mosquito is a threat to human health across the globe. The A. aegypti genome was recently re-sequenced and re-assembled. Due to a combination of long-read PacBio and Hi-C sequencing, the AaegL5 assembly is chromosome complete and significantly improves the assembly in key areas such as the M/m sex-determining locus. Release of the updated genome assembly has precipitated the need to reprocess historical functional genomic data sets, including cis-regulatory element (CRE) maps that had previously been generated for A. aegypti. RESULTS: We re-processed and re-analyzed the A. aegypti whole embryo FAIRE seq data to create an updated embryonic CRE map for the AaegL5 genome. We validated that the new CRE map recapitulates key features of the original AaegL3 CRE map. Further, we built on the improved assembly in the M/m locus to analyze overlaps of open chromatin regions with genes. To support the validation, we created a new method (PeakMatcher) for matching peaks from the same experimental data set across genome assemblies. CONCLUSION: Use of PeakMatcher software, which is available publicly under an open-source license, facilitated the release of an updated and validated CRE map, which is available through the NIH GEO. These findings demonstrate that PeakMatcher software will be a useful resource for validation and transferring of previous annotations to updated genome assemblies.

Perspectives on Gene Regulatory Network Evolution.

Animal development proceeds through the activity of genes and their cis-regulatory modules (CRMs) working together in sets of gene regulatory networks (GRNs). The emergence of species-specific traits and novel structures results from evolutionary changes in GRNs. Recent work in a wide variety of animal models, and particularly in insects, has started to reveal the modes and mechanisms of GRN evolution. I discuss here various aspects of GRN evolution and argue that developmental system drift (DSD), in which conserved phenotype is nevertheless a result of changed genetic interactions, should regularly be viewed from the perspective of GRN evolution. Advances in methods to discover related CRMs in diverse insect species, a critical requirement for detailed GRN characterization, are also described.

How to study enhancers in non-traditional insect models.

Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue- and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of Drosophila have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as Drosophila, we focus on a subset of computational and experimental approaches that are likely applicable to non-Drosophila insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.

Computational enhancer prediction: evaluation and improvements.

BACKGROUND: Identifying transcriptional enhancers and other cis-regulatory modules (CRMs) is an important goal of post-sequencing genome annotation. Computational approaches provide a useful complement to empirical methods for CRM discovery, but it is critical that we develop effective means to evaluate their performance in terms of estimating their sensitivity and specificity. RESULTS: We introduce here pCRMeval, a pipeline for in silico evaluation of any enhancer prediction tools that are flexible enough to be applied to the Drosophila melanogaster genome. pCRMeval compares the result of predictions with the extensive existing knowledge of experimentally-validated Drosophila CRMs in order to estimate the precision and relative sensitivity of the prediction method. In the case of supervised prediction methods-when training data composed of validated CRMs are used-pCRMeval can also assess the sensitivity of specific training sets. We demonstrate the utility of pCRMeval through evaluation of our SCRMshaw CRM prediction method and training data. By measuring the impact of different parameters on SCRMshaw performance, as assessed by pCRMeval, we develop a more robust version of SCRMshaw, SCRMshaw_HD, that improves the number of predictions while maintaining sensitivity and specificity. Our analysis also demonstrates that SCRMshaw_HD, when applied to increasingly less well-assembled genomes, maintains its strong predictive power with only a minor drop-off in performance. CONCLUSION: Our pCRMeval pipeline provides a general framework for evaluation that can be applied to any CRM prediction method, particularly a supervised method. While we make use of it here primarily to test and improve a particular method for CRM prediction, SCRMshaw, pCRMeval should provide a valuable platform to the research community not only for evaluating individual methods, but also for comparing between competing methods.

Redeployment of a conserved gene regulatory network during Aedes aegypti development.

Changes in gene regulatory networks (GRNs) underlie the evolution of morphological novelty and developmental system drift. The fruitfly Drosophila melanogaster and the dengue and Zika vector mosquito Aedes aegypti have substantially similar nervous system morphology. Nevertheless, they show significant divergence in a set of genes co-expressed in the midline of the Drosophila central nervous system, including the master regulator single minded and downstream genes including short gastrulation, Star, and NetrinA. In contrast to Drosophila, we find that midline expression of these genes is either absent or severely diminished in A. aegypti. Instead, they are co-expressed in the lateral nervous system. This suggests that in A. aegypti this "midline GRN" has been redeployed to a new location while lost from its previous site of activity. In order to characterize the relevant GRNs, we employed the SCRMshaw method we previously developed to identify transcriptional cis-regulatory modules in both species. Analysis of these regulatory sequences in transgenic Drosophila suggests that the altered gene expression observed in A. aegypti is the result of trans-dependent redeployment of the GRN, potentially stemming from cis-mediated changes in the expression of sim and other as-yet unidentified regulators. Our results illustrate a novel "repeal, replace, and redeploy" mode of evolution in which a conserved GRN acquires a different function at a new site while its original function is co-opted by a different GRN. This represents a striking example of developmental system drift in which the dramatic shift in gene expression does not result in gross morphological changes, but in more subtle differences in development and function of the late embryonic nervous system.

A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species.

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Improved methodology for fixation and preparation of Aedes aegypti embryos.

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.

Improved accuracy of supervised CRM discovery with interpolated Markov models and cross-species comparison.

Despite recent advances in experimental approaches for identifying transcriptional cis-regulatory modules (CRMs, 'enhancers'), direct empirical discovery of CRMs for all genes in all cell types and environmental conditions is likely to remain an elusive goal. Effective methods for computational CRM discovery are thus a critically needed complement to empirical approaches. However, existing computational methods that search for clusters of putative binding sites are ineffective if the relevant TFs and/or their binding specificities are unknown. Here, we provide a significantly improved method for 'motif-blind' CRM discovery that does not depend on knowledge or accurate prediction of TF-binding motifs and is effective when limited knowledge of functional CRMs is available to 'supervise' the search. We propose a new statistical method, based on 'Interpolated Markov Models', for motif-blind, genome-wide CRM discovery. It captures the statistical profile of variable length words in known CRMs of a regulatory network and finds candidate CRMs that match this profile. The method also uses orthologs of the known CRMs from closely related genomes. We perform in silico evaluation of predicted CRMs by assessing whether their neighboring genes are enriched for the expected expression patterns. This assessment uses a novel statistical test that extends the widely used Hypergeometric test of gene set enrichment to account for variability in intergenic lengths. We find that the new CRM prediction method is superior to existing methods. Finally, we experimentally validate 12 new CRM predictions by examining their regulatory activity in vivo in Drosophila; 10 of the tested CRMs were found to be functional, while 6 of the top 7 predictions showed the expected activity patterns. We make our program available as downloadable source code, and as a plugin for a genome browser installed on our servers.

REDfly v3.0: toward a comprehensive database of transcriptional regulatory elements in Drosophila.

The REDfly database of Drosophila transcriptional cis-regulatory elements provides the broadest and most comprehensive available resource for experimentally validated cis-regulatory modules and transcription factor binding sites among the metazoa. The third major release of the database extends the utility of REDfly as a powerful tool for both computational and experimental studies of transcription regulation. REDfly v3.0 includes the introduction of new data classes to expand the types of regulatory elements annotated in the database along with a roughly 40% increase in the number of records. A completely redesigned interface improves access for casual and power users alike; among other features it now automatically provides graphical views of the genome, displays images of reporter gene expression and implements improved capabilities for database searching and results filtering. REDfly is freely accessible at http://redfly.ccr.buffalo.edu.

Evaluation of metric and representation learning approaches: Effects of representations driven by relative distance on the performance.

Several deep neural network architectures have emerged recently for metric learning. We asked which architecture is the most effective in measuring the similarity or dissimilarity among images. To this end, we evaluated six networks on a standard image set. We evaluated variational autoencoders, Siamese networks, triplet networks, and variational auto-encoders combined with Siamese or triplet networks. These networks were compared to a baseline network consisting of multiple separable convolutional layers. Our study revealed the following: (i) the triplet architecture proved the most effective one due to learning a relative distance - not an absolute distance; (ii) combining auto-encoders with networks that learn metrics (e.g., Siamese or triplet networks) is unwarranted; and (iii) an architecture based on separable convolutional layers is a reasonable simple alternative to triplet networks. These results can potentially impact our field by encouraging architects to develop advanced networks that take advantage of separable convolution and relative distance.