Drosophila immune priming to Enterococcus faecalis relies on immune tolerance rather than resistance.

Innate immune priming increases an organism's survival of a second infection after an initial, non-lethal infection. We used Drosophila melanogaster and an insect-derived strain of Enterococcus faecalis to study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreased E.

Shadow enhancers mediate trade-offs between transcriptional noise and fidelity.

Enhancers are stretches of regulatory DNA that bind transcription factors (TFs) and regulate the expression of a target gene. Shadow enhancers are two or more enhancers that regulate the same target gene in space and time and are associated with most animal developmental genes. These multi-enhancer systems can drive more consistent transcription than single enhancer systems.

Longitudinal monitoring of individual infection progression in .

The innate immune system is critical for infection survival. is a key model for understanding the evolution and dynamics of innate immunity. Current toolsets for fly infection studies are limited in throughput and, because of their destructive nature, cannot generate longitudinal measurements in individual animals.

Two promoters integrate multiple enhancer inputs to drive wild-type knirps expression in the Drosophila melanogaster embryo.

Proper development depends on precise spatiotemporal gene expression patterns. Most developmental genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. We hypothesize that multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism.

Mechanistic gene networks inferred from single-cell data with an outlier-insensitive method.

With advances in single-cell techniques, measuring gene dynamics at cellular resolution has become practicable. In contrast, the increased complexity of data has made it more challenging computationally to unravel underlying biological mechanisms. Thus, it is critical to develop novel computational methods capable of dealing with such complexity and of providing predictive deductions from such data.

Molecular competition can shape enhancer activity in the embryo.

Transgenic reporters allow the measurement of regulatory DNA activity and consequently have long been useful tools for studying enhancers. Despite their utility, few studies have investigated the effects these reporters may have on the expression of other genes. Understanding these effects is required to accurately interpret reporter data and characterize gene regulatory mechanisms.

The mode of expression divergence in fat body is infection-specific.

Transcription is controlled by interactions of -acting DNA elements with diffusible -acting factors. Changes in or factors can drive expression divergence within and between species, and their relative prevalence can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole-body expression measurements in one condition.

Enhancer redundancy in development and disease.

Shadow enhancers are seemingly redundant transcriptional cis-regulatory elements that regulate the same gene and drive overlapping expression patterns. Recent studies have shown that shadow enhancers are remarkably abundant and control most developmental gene expression in both invertebrates and vertebrates, including mammals. Shadow enhancers might provide an important mechanism for buffering gene expression against mutations in non-coding regulatory regions of genes implicated in human disease.

A Mutation in the Stripe 2 Minimal Enhancer Is Buffered by Flanking Sequences.

Enhancers are DNA sequences composed of transcription factor binding sites that drive complex patterns of gene expression in space and time. Until recently, studying enhancers in their genomic context was technically challenging. Therefore, minimal enhancers, the shortest pieces of DNA that can drive an expression pattern that resembles a gene's endogenous pattern, are often used to study features of enhancer function.

Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic.

Shadow enhancers, groups of seemingly redundant enhancers, are found in a wide range of organisms and are critical for robust developmental patterning. However, their mechanism of action is unknown. We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor (TF) inputs at the individual enhancers.

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

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.

Quantitative Comparison of the Anterior-Posterior Patterning System in the Embryos of Five Species.

Complex spatiotemporal gene expression patterns direct the development of the fertilized egg into an adult animal. Comparisons across species show that, in spite of changes in the underlying regulatory DNA sequence, developmental programs can be maintained across millions of years of evolution. Reciprocally, changes in gene expression can be used to generate morphological novelty.

Inference of Transcription Factor Regulation Patterns Using Gene Expression Covariation in Natural Populations of .

Gene regulatory networks control the complex programs that drive development. Deciphering the connections between transcription factors (TFs) and target genes is challenging, in part because TFs bind to thousands of places in the genome but control expression through a subset of these binding events. We hypothesize that we can combine natural variation of expression levels and predictions of TF binding sites to identify TF targets.

Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos.

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.

Analysis of Genetic Variation Indicates DNA Shape Involvement in Purifying Selection.

Noncoding DNA sequences, which play various roles in gene expression and regulation, are under evolutionary pressure. Gene regulation requires specific protein-DNA binding events, and our previous studies showed that both DNA sequence and shape readout are employed by transcription factors (TFs) to achieve DNA binding specificity. By investigating the shape-disrupting properties of single nucleotide polymorphisms (SNPs) in human regulatory regions, we established a link between disruptive local DNA shape changes and loss of specific TF binding.

Quantitative Measurement and Thermodynamic Modeling of Fused Enhancers Support a Two-Tiered Mechanism for Interpreting Regulatory DNA.

Computational models of enhancer function generally assume that transcription factors (TFs) exert their regulatory effects independently, modeling an enhancer as a "bag of sites." These models fail on endogenous loci that harbor multiple enhancers, and a "two-tier" model appears better suited: in each enhancer TFs work independently, and the total expression is a weighted sum of their expression readouts. Here, we test these two opposing views on how cis-regulatory information is integrated.

An Enhancer's Length and Composition Are Shaped by Its Regulatory Task.

Enhancers drive the gene expression patterns required for virtually every process in metazoans. We propose that enhancer length and transcription factor (TF) binding site composition-the number and identity of TF binding sites-reflect the complexity of the enhancer's regulatory task. In development, we define regulatory task complexity as the number of fates specified in a set of cells at once.

SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites.

Krüppel Expression Levels Are Maintained through Compensatory Evolution of Shadow Enhancers.

Many developmental genes are controlled by shadow enhancers—pairs of enhancers that drive overlapping expression patterns. We hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. To test this hypothesis, we analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos.

Yearly planning meetings: individualized development plans aren't just more paperwork.

The National Institutes of Health (NIH) encourages trainees to make Individualized Development Plans to help them prepare for academic and nonacademic careers. We describe our approach to building an Individualized Development Plan, the reasons we find them useful and empowering for both PIs and trainees, and resources to help other labs implement them constructively.

A gene expression atlas of a bicoid-depleted Drosophila embryo reveals early canalization of cell fate.

In developing embryos, gene regulatory networks drive cells towards discrete terminal fates, a process called canalization. We studied the behavior of the anterior-posterior segmentation network in Drosophila melanogaster embryos by depleting a key maternal input, bicoid (bcd), and measuring gene expression patterns of the network at cellular resolution. This method results in a gene expression atlas containing the levels of mRNA or protein expression of 13 core patterning genes over six time points for every cell of the blastoderm embryo.