Chromatin enables precise and scalable gene regulation with factors of limited specificity.

Biophysical constraints limit the specificity with which transcription factors (TFs) can target regulatory DNA. While individual nontarget binding events may be low affinity, the sheer number of such interactions could present a challenge for gene regulation by degrading its precision or possibly leading to an erroneous induction state. Chromatin can prevent nontarget binding by rendering DNA physically inaccessible to TFs, at the cost of energy-consuming remodeling orchestrated by pioneer factors (PFs).

Using AI to prevent the insect apocalypse: toward new environmental risk assessment procedures.

Insect populations are declining globally, with multiple potential drivers identified. However, experimental data are needed to understand their relative contributions. We highlight the sublethal effects of pesticides at field-relevant concentrations, often overlooked in standard environmental risk assessments (ERA), as significant contributors to these declines.

Pervasive sublethal effects of agrochemicals on insects at environmentally relevant concentrations.

Insect biomass is declining globally, likely driven by climate change and pesticide use, yet systematic studies on the effects of various chemicals remain limited. In this work, we used a chemical library of 1024 molecules-covering insecticides, herbicides, fungicides, and plant growth inhibitors-to assess the impact of sublethal pesticide doses on insects. In , 57% of chemicals affected larval behavior, and a higher proportion compromised long-term survivability.

TF-High-Evolutionary: In Vivo Mutagenesis of Gene Regulatory Networks for the Study of the Genetics and Evolution of the Drosophila Regulatory Genome.

Understanding the evolutionary potential of mutations in gene regulatory networks is essential to furthering the study of evolution and development. However, in multicellular systems, genetic manipulation of regulatory networks in a targeted and high-throughput way remains challenging. In this study, we designed TF-High-Evolutionary (HighEvo), a transcription factor (TF) fused with a base editor (activation-induced deaminase), to continuously induce germline mutations at TF-binding sites across regulatory networks in Drosophila.

LarvaTagger: manual and automatic tagging of Drosophila larval behaviour.

Motivation: As more behavioural assays are carried out in large-scale experiments on Drosophila larvae, the definitions of the archetypal actions of a larva are regularly refined. In addition, video recording and tracking technologies constantly evolve. Consequently, automatic tagging tools for Drosophila larval behaviour must be retrained to learn new representations from new data.

Rapid response of fly populations to gene dosage across development and generations.

Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network's regulatory outputs and a decrease in fitness.

Emerging questions in transcriptional regulation.

What new questions can we ask about transcriptional regulation given recent developments in large-scale approaches?

Mutational scans reveal differential evolvability of promoters and enhancers.

Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations.

Enhancer architecture and chromatin accessibility constrain phenotypic space during Drosophila development.

Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern.

Developmental phenomics suggests that H3K4 monomethylation confers multi-level phenotypic robustness.

How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. Here, we genetically reduce H3K4me1 deposition in Drosophila melanogaster and find that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness.

A synthetic synthesis to explore animal evolution and development.

Identifying the general principles by which genotypes are converted into phenotypes remains a challenge in the post-genomic era. We still lack a predictive understanding of how genes shape interactions among cells and tissues in response to signalling and environmental cues, and hence how regulatory networks generate the phenotypic variation required for adaptive evolution. Here, we discuss how techniques borrowed from synthetic biology may facilitate a systematic exploration of evolvability across biological scales.

Nuclear morphogenesis: forming a heterogeneous nucleus during embryogenesis.

An embryo experiences increasingly complex spatial and temporal patterns of gene expression as it matures, guiding the morphogenesis of its body. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed that the nuclear distributions of transcription factors and histone modifications undergo a similar transformation of increasing heterogeneity. This spatial partitioning of the nucleus could lead to distinct local regulatory environments in space and time that are tuned for specific genes.

An open-source semi-automated robotics pipeline for embryo immunohistochemistry.

A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos.

Dense and pleiotropic regulatory information in a developmental enhancer.

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster.

Robust and efficient gene regulation through localized nuclear microenvironments.

Developmental enhancers drive gene expression in specific cell types during animal development. They integrate signals from many different sources mediated through the binding of transcription factors, producing specific responses in gene expression. Transcription factors often bind low-affinity sequences for only short durations.

Enhancer-Promoter Communication: Thinking Outside the TAD.

How does the folding of the genome relate to the regulation of gene expression? Using fly embryos and quantitative live imaging, a recent study by Yokoshi et al. reveals that the answer might depend on whether enhancer-promoter communication occurs inside or in-between topological domains.

Multi-enhancer transcriptional hubs confer phenotypic robustness.

We previously showed in embryos that low-affinity Ultrabithorax (Ubx)-responsive () enhancers drive expression using localized transcriptional environments and that active enhancers on different chromosomes tended to colocalize (Tsai et al., 2017). Here, we test the hypothesis that these multi-enhancer 'hubs' improve phenotypic resilience to stress by buffering against decreases in transcription factor concentrations and transcriptional output.

Transvection Goes Live-Visualizing Enhancer-Promoter Communication between Chromosomes.

Lim et al. (2018) use live imaging in Drosophila embryos to show that enhancers can drive transcription from promoters on another chromosome when they are in close proximity. In addition, they show that multiple promoters can access the same enhancer without competition, potentially sharing a pool of factors in a transcriptional "hub.

Accurate and sensitive quantification of protein-DNA binding affinity.

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites.

Nuclear microenvironments modulate transcription from low-affinity enhancers.

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the () locus and related enhancers in nuclear microenvironments of high Ubx concentrations.

Using synthetic biology to study gene regulatory evolution.

Transcriptional enhancers specify the precise time, level, and location of gene expression. Disentangling and characterizing the components of enhancer activity in multicellular eukaryotic development has proven challenging because enhancers contain activator and repressor binding sites for multiple factors that each exert nuanced, context-dependent control of enhancer activity. Recent advances in synthetic biology provide an almost unlimited ability to create and modify regulatory elements and networks, offering unprecedented power to study gene regulation.

Functional regulatory evolution outside of the minimal stripe 2 enhancer.

Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. Although many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called 'minimal' enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here, we explore how the () locus has evolved by testing its activity in the divergent genome.