Raquel Marco-Ferreres's research while affiliated with European Molecular Biology Laboratory and other places

To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer–promoter (E–P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E–P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E–P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E–P proximity reflect changes in activity. The mode of E–P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E–P proximity is coupled to activation.

Chromatin loops between gene pairs have been observed in diverse contexts in both flies and vertebrates. Combining high-resolution Capture-C, DNA fluorescence in situ hybridization, and genetic perturbations, we dissect the functional role of three loops between genes with related function during Drosophila embryogenesis. By mutating the loop anchor (but not the gene) or the gene (but not loop anchor), we disentangle loop formation and gene expression and show that the 3D proximity of paralogous gene loci supports their co-regulation. Breaking the loop leads to either an attenuation or enhancement of expression and perturbs their relative levels of expression and cross-regulation. Although many loops appear constitutive across embryogenesis, their function can change in different developmental contexts. Taken together, our results indicate that chromatin gene-gene loops act as architectural scaffolds that can be used in different ways in different contexts to fine-tune the coordinated expression of genes with related functions and sustain their cross-regulation.

To regulate gene expression, enhancers must come into proximity with their target gene. At some loci the timing of enhancer-promoter proximity is uncoupled from gene activation, while at others it is tightly linked. Here, we assessed this more globally for 600 characterized enhancers or promoters (E/P) with tissue-specific activity in Drosophila embryos, by performing Capture-C and insulator ChIP in FACS-purified myogenic or neurogenic cells at different stages of embryogenesis. This high-resolution view enabled direct comparison between E/P interactions and activity across 5 developmental conditions. This revealed largely invariant E/P contacts between the blastoderm and cell fate specification stages, despite changes in activity. However, E/P interactions diverge during terminal tissue differentiation when many tissue-specific interactions are gained on top of a pre-existing topology. Changes in E/P proximity reflect changes in enhancer activity and gene activation, and are generally not accompanied by changes in insulator binding. Using transgenes and deletions, we show that many tissue-specific interactions represent functional E-P pairs. Our results reveal a shift in E-P landscapes as embryogenesis proceeds, from largely pre-formed topologies at early stages to more distal tissue-specific loops during differentiation, when E/P proximity appears coupled to activation.

Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY’s efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.

Enhancers are essential drivers of cell states, yet the relationship between accessibility, regulatory activity, and in vivo lineage commitment during embryogenesis remains poorly understood. Here, we measure chromatin accessibility in isolated neural and mesodermal lineages across a time course of Drosophila embryogenesis. Promoters, including tissue-specific genes, are often constitutively open, even in contexts where the gene is not expressed. In contrast, the majority of distal elements have dynamic, tissue-specific accessibility. Enhancer priming appears rarely within a lineage, perhaps reflecting the speed of Drosophila embryogenesis. However, many tissue-specific enhancers are accessible in other lineages early on and become progressively closed as embryogenesis proceeds. We demonstrate the usefulness of this tissue- and time-resolved resource to definitively identify single-cell clusters, to uncover predictive motifs, and to identify many regulators of tissue development. For one such predicted neural regulator, l(3)neo38, we generate a loss-of-function mutant and uncover an essential role for neuromuscular junction and brain development.

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts. Here, we applied a machine learning approach by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatio-temporal predictions for all genes. We developed structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. We systematically evaluated our performance via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. We further show the potential of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at our webserver http://find.princeton.edu, which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles.