Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation.

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.

Chromatin gene-gene loops support the cross-regulation of genes with related function.

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.

Targeted design of synthetic enhancers for selected tissues in the Drosophila embryo.

Enhancers control gene expression and have crucial roles in development and homeostasis. However, the targeted de novo design of enhancers with tissue-specific activities has remained challenging. Here we combine deep learning and transfer learning to design tissue-specific enhancers for five tissues in the Drosophila melanogaster embryo: the central nervous system, epidermis, gut, muscle and brain.

CTCF, BEAF-32, and CP190 are not required for the establishment of TADs in early embryos but have locus-specific roles.

The boundaries of topologically associating domains (TADs) are delimited by insulators and/or active promoters; however, how they are initially established during embryogenesis remains unclear. Here, we examined this during the first hours of embryogenesis. DNA-FISH confirms that intra-TAD pairwise proximity is established during zygotic genome activation (ZGA) but with extensive cell-to-cell heterogeneity.

Enhancers have more cofactor specificity than we think: A potential new way to classify enhancers based on their functional cofactor requirements.

By systematically assessing the effects of depleting eight cofactors on enhancer activity, Neumayr et al. (2022) found that different enhancers have different requirements for some perceived universal cofactors. While some cofactors influence enhancer strength, others affect enhancer-promoter specificity.

The continuum of embryonic development at single-cell resolution.

is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time.

Regulating specificity in enhancer-promoter communication.

Enhancers are cis-regulatory elements that can activate transcription remotely to regulate a specific pattern of a gene's expression. Genes typically have many enhancers that are often intermingled in the loci of other genes. To regulate expression, enhancers must therefore activate their correct promoter while ignoring others that may be in closer linear proximity.

Simultaneous cellular and molecular phenotyping of embryonic mutants using single-cell regulatory trajectories.

Developmental progression and cellular diversity are largely driven by transcription factors (TFs); yet, characterizing their loss-of-function phenotypes remains challenging and often disconnected from their underlying molecular mechanisms. Here, we combine single-cell regulatory genomics with loss-of-function mutants to jointly assess both cellular and molecular phenotypes. Performing sci-ATAC-seq at eight overlapping time points during Drosophila mesoderm development could reconstruct the developmental trajectories of all major muscle types and reveal the TFs and enhancers involved.

scDALI: modeling allelic heterogeneity in single cells reveals context-specific genetic regulation.

While it is established that the functional impact of genetic variation can vary across cell types and states, capturing this diversity remains challenging. Current studies using bulk sequencing either ignore this heterogeneity or use sorted cell populations, reducing discovery and explanatory power. Here, we develop scDALI, a versatile computational framework that integrates information on cellular states with allelic quantifications of single-cell sequencing data to characterize cell-state-specific genetic effects.

Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.

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.

The hourglass model of evolutionary conservation during embryogenesis extends to developmental enhancers with signatures of positive selection.

Inter-species comparisons of both morphology and gene expression within a phylum have revealed a period in the middle of embryogenesis with more similarity between species compared with earlier and later time points. This "developmental hourglass" pattern has been observed in many phyla, yet the evolutionary constraints on gene expression, as well as the underlying mechanisms of how this is regulated, remain elusive. Moreover, the role of positive selection on gene regulation in the more diverged earlier and later stages of embryogenesis remains unknown.

Chromatin accessibility profiling methods.

Chromatin accessibility, or the physical access to chromatinized DNA, is a widely studied characteristic of the eukaryotic genome. As active regulatory DNA elements are generally 'accessible', the genome-wide profiling of chromatin accessibility can be used to identify candidate regulatory genomic regions in a tissue or cell type. Multiple biochemical methods have been developed to profile chromatin accessibility, both in bulk and at the single-cell level.

-acting variation is common across regulatory layers but is often buffered during embryonic development.

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequences, and chromatin. How DNA mutations affecting any one of these regulatory "layers" are buffered or propagated to gene expression remains unclear. To address this, we quantified allele-specific changes in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight crosses at three embryonic stages, yielding a comprehensive data set of 240 samples spanning multiple regulatory layers.

Lineage-Resolved Enhancer and Promoter Usage during a Time Course of Embryogenesis.

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.

The road ahead in genetics and genomics.

In celebration of the 20th anniversary of Nature Reviews Genetics, we asked 12 leading researchers to reflect on the key challenges and opportunities faced by the field of genetics and genomics. Keeping their particular research area in mind, they take stock of the current state of play and emphasize the work that remains to be done over the next few years so that, ultimately, the benefits of genetic and genomic research can be felt by everyone.

Characterization of Button Loci that Promote Homologous Chromosome Pairing and Cell-Type-Specific Interchromosomal Gene Regulation.

Homologous chromosomes colocalize to regulate gene expression in processes including genomic imprinting, X-inactivation, and transvection. In Drosophila, homologous chromosomes pair throughout development, promoting transvection. The "button" model of pairing proposes that specific regions along chromosomes pair with high affinity.

Chromatin topology, condensates and gene regulation: shifting paradigms or just a phase?

In the past decade, two major advances in our understanding of nuclear organization have taken the field of gene regulation by storm. First, technologies that can analyze the three-dimensional conformation of chromatin have revealed how the genome is organized and have provided novel insights into how regulatory regions in the genome interact. Second, the recognition that many proteins can form membraneless compartments through liquid-liquid phase separation (LLPS) has challenged long-standing notions of how proteins within the nucleus are organized and has offered a tantalizing general mechanism by which many aspects of nuclear function may be regulated.

Accurate genome-wide predictions of spatio-temporal gene expression during embryonic 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.

Highly rearranged chromosomes reveal uncoupling between genome topology and gene expression.

Chromatin topology is intricately linked to gene expression, yet its functional requirement remains unclear. Here, we comprehensively assessed the interplay between genome topology and gene expression using highly rearranged chromosomes (balancers) spanning ~75% of the Drosophila genome. Using transheterozyte (balancer/wild-type) embryos, we measured allele-specific changes in topology and gene expression in cis, while minimizing trans effects.

The Role of Chromatin Accessibility in cis-Regulatory Evolution.

Transcription factor (TF) binding is determined by sequence as well as chromatin accessibility. Although the role of accessibility in shaping TF-binding landscapes is well recorded, its role in evolutionary divergence of TF binding, which in turn can alter cis-regulatory activities, is not well understood. In this work, we studied the evolution of genome-wide binding landscapes of five major TFs in the core network of mesoderm specification, between Drosophila melanogaster and Drosophila virilis, and examined its relationship to accessibility and sequence-level changes.

Expanding the mesodermal transcriptional network by genome-wide identification of Zinc finger homeodomain 1 (Zfh1) targets.

The Drosophila transcription factor (TF) Zfh1 has distinct roles compared to the cell lineage-determining TFs in almost all mesoderm-derived tissues. Here, we link Zfh1 to the well-characterized mesodermal transcriptional network. We identify five enhancers integrating upstream regulatory inputs from mesodermal TFs and directing zfh1 expression in mesoderm.

The role of transcription in shaping the spatial organization of the genome.

The spatial organization of the genome into compartments and topologically associated domains can have an important role in the regulation of gene expression. But could gene expression conversely regulate genome organization? Here, we review recent studies that assessed the requirement of transcription and/or the transcription machinery for the establishment or maintenance of genome topology. The results reveal different requirements at different genomic scales.

Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis.

Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. Here, we performed a tailored investigation of lncRNA expression and function during Drosophila embryogenesis, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. Our results almost double the number of annotated lncRNAs expressed at these embryonic stages.

Developmental enhancers and chromosome topology.

Developmental enhancers mediate on/off patterns of gene expression in specific cell types at particular stages during metazoan embryogenesis. They typically integrate multiple signals and regulatory determinants to achieve precise spatiotemporal expression. Such enhancers can map quite far-one megabase or more-from the genes they regulate.

The Insulator Protein CTCF Is Required for Correct Gene Expression, but Not for Embryonic Development in .

Insulator binding proteins (IBPs) play an important role in regulating gene expression by binding to specific DNA sites to facilitate appropriate gene regulation. There are several IBPs in , each defined by their ability to insulate target gene promoters in transgenic assays from the activating or silencing effects of neighboring regulatory elements. Of these, only CCCTC-binding factor (CTCF) has an obvious ortholog in mammals.