Evolutionary analyses of intrinsically disordered regions reveal widespread signals of conservation.

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 7,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns.

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

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Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos.

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the use of single-nucleus RNA sequencing in Drosophila embryos prior to cellularization, here we look at gene expression in control and insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we provide a resource of candidate differentially expressed genes that might show local changes in gene expression between control and maternal dCTCF null nuclei with no detectable differential expression in bulk. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo.

Impact of essential workers in the context of social distancing for epidemic control.

New emerging infectious diseases are identified every year, a subset of which become global pandemics like COVID-19. In the case of COVID-19, many governments have responded to the ongoing pandemic by imposing social policies that restrict contacts outside of the home, resulting in a large fraction of the workforce either working from home or not working. To ensure essential services, however, a substantial number of workers are not subject to these limitations, and maintain many of their pre-intervention contacts. To explore how contacts among such "essential" workers, and between essential workers and the rest of the population, impact disease risk and the effectiveness of pandemic control, we evaluated several mathematical models of essential worker contacts within a standard epidemiology framework. The models were designed to correspond to key characteristics of cashiers, factory employees, and healthcare workers. We find in all three models that essential workers are at substantially elevated risk of infection compared to the rest of the population, as has been documented, and that increasing the numbers of essential workers necessitates the imposition of more stringent controls on contacts among the rest of the population to manage the pandemic. Importantly, however, different archetypes of essential workers differ in both their individual probability of infection and impact on the broader pandemic dynamics, highlighting the need to understand and target intervention for the specific risks faced by different groups of essential workers. These findings, especially in light of the massive human costs of the current COVID-19 pandemic, indicate that contingency plans for future epidemics should account for the impacts of essential workers on disease spread.

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A phylogeny for the Drosophila montium species group: A model clade for comparative analyses.

The Drosophila montium species group is a clade of 94 named species, closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. We use genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of our phylogenetic inferences, we use 3 non-overlapping sets of 20 single-copy coding sequences and analyze all 60 genes with both Bayesian and maximum likelihood methods. Our analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, our analyses also support the monophyly of all seven subgroups proposed within the montium group. Our phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. We discuss methodological issues that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade.

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene.

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high-temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shapes complex developmental patterns.

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Whole Genome Sequences of 23 Species from the Drosophila montium Species Group (Diptera: Drosophilidae): A Resource for Testing Evolutionary Hypotheses.

Large groups of species with well-defined phylogenies are excellent systems for testing evolutionary hypotheses. In this paper, we describe the creation of a comparative genomic resource consisting of 23 genomes from the species-rich Drosophila montium species group, 22 of which are presented here for the first time. The montium group is well-positioned for clade genomics. Within the montium clade, evolutionary distances are such that large numbers of sequences can be accurately aligned while also recovering strong signals of divergence; and the distance between the montium group and D. melanogaster is short enough so that orthologous sequence can be readily identified. All genomes were assembled from a single, small-insert library using MaSuRCA, before going through an extensive post-assembly pipeline. Estimated genome sizes within the montium group range from 155 Mb to 223 Mb (mean = 196 Mb). The absence of long-distance information during the assembly process resulted in fragmented assemblies, with the scaffold NG50s varying widely based on repeat content and sample heterozygosity (min = 18 kb, max = 390 kb, mean = 74 kb). The total scaffold length for most assemblies is also shorter than the estimated genome size, typically by 5-15%. However, subsequent analysis showed that our assemblies are highly complete. Despite large differences in contiguity, all assemblies contain at least 96% of known single-copy Dipteran genes (BUSCOs, n = 2,799). Similarly, by aligning our assemblies to the D. melanogaster genome and remapping coordinates for a large set of transcriptional enhancers (n = 3,457), we showed that each montium assembly contains orthologs for at least 91% of D. melanogaster enhancers. Importantly, the genic and enhancer contents of our assemblies are comparable to that of far more contiguous Drosophila assemblies. The alignment of our own D. serrata assembly to a previously published PacBio D. serrata assembly also showed that our longest scaffolds (up to 1 Mb) are free of large-scale misassemblies. Our genome assemblies are a valuable resource that can be used to further resolve the montium group phylogeny; study the evolution of protein-coding genes and cis-regulatory sequences; and determine the genetic basis of ecological and behavioral adaptations.

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos.

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy.

In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.

Gene family innovation, conservation and loss on the animal stem lineage.

Choanoflagellates, the closest living relatives of animals, can provide unique insights into the changes in gene content that preceded the origin of animals. However, only two choanoflagellate genomes are currently available, providing poor coverage of their diversity. We sequenced transcriptomes of 19 additional choanoflagellate species to produce a comprehensive reconstruction of the gains and losses that shaped the ancestral animal gene repertoire. We identified ~1944 gene families that originated on the animal stem lineage, of which only 39 are conserved across all animals in our study. In addition, ~372 gene families previously thought to be animal-specific, including Notch, Delta, and homologs of the animal Toll-like receptor genes, instead evolved prior to the animal-choanoflagellate divergence. Our findings contribute to an increasingly detailed portrait of the gene families that defined the biology of the Urmetazoan and that may underpin core features of extant animals.

Convergent evolution of gene expression in two high-toothed stickleback populations.

Changes in developmental gene regulatory networks enable evolved changes in morphology. These changes can be in cis regulatory elements that act in an allele-specific manner, or changes to the overall trans regulatory environment that interacts with cis regulatory sequences. Here we address several questions about the evolution of gene expression accompanying a convergently evolved constructive morphological trait, increases in tooth number in two independently derived freshwater populations of threespine stickleback fish (Gasterosteus aculeatus). Are convergently evolved cis and/or trans changes in gene expression associated with convergently evolved morphological evolution? Do cis or trans regulatory changes contribute more to gene expression changes accompanying an evolved morphological gain trait? Transcriptome data from dental tissue of ancestral low-toothed and two independently derived high-toothed stickleback populations revealed significantly shared gene expression changes that have convergently evolved in the two high-toothed populations. Comparing cis and trans regulatory changes using phased gene expression data from F1 hybrids, we found that trans regulatory changes were predominant and more likely to be shared among both high-toothed populations. In contrast, while cis regulatory changes have evolved in both high-toothed populations, overall these changes were distinct and not shared among high-toothed populations. Together these data suggest that a convergently evolved trait can occur through genetically distinct regulatory changes that converge on similar trans regulatory environments.

The ecology of the Drosophila-yeast mutualism in wineries.

The fruit fly, Drosophila melanogaster, is preferentially found on fermenting fruits. The yeasts that dominate the microbial communities of these substrates are the primary food source for developing D. melanogaster larvae, and adult flies manifest a strong olfactory system-mediated attraction for the volatile compounds produced by these yeasts during fermentation. Although most work on this interaction has focused on the standard laboratory yeast Saccharomyces cerevisiae, a wide variety of other yeasts naturally ferment fallen fruit. Here we address the open question of whether D. melanogaster preferentially associates with distinct yeasts in different, closely-related environments. We characterized the spatial and temporal dynamics of Drosophila-associated fungi in Northern California wineries that use organic grapes and natural fermentation using high-throughput, short-amplicon sequencing. We found that there is nonrandom structure in the fungal communities that are vectored by flies both between and within vineyards. Within wineries, the fungal communities associated with flies in cellars, fermentation tanks, and pomace piles are distinguished by varying abundances of a small number of yeast species. To investigate the origins of this structure, we assayed Drosophila attraction to, oviposition on, larval development in, and longevity when consuming the yeasts that distinguish vineyard microhabitats from each other. We found that wild fly lines did not respond differentially to the yeast species that distinguish winery habitats in habitat specific manner. Instead, this subset of yeast shares traits that make them attractive to and ensure their close association with Drosophila.

Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo.

As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However it remains unclear how regions of activity are established during early embryogenesis, and if they are the product of spatially restricted or ubiquitous processes. To shed light on this question, we probed chromatin accessibility across the anterior-posterior axis (A-P) of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is highly similar between the two halves, with regions that manifest significant accessibility in one half of the embryo almost always accessible in the other half, even for promoters that are active in exclusively one half of the embryo. These data support previous studies that show that chromatin accessibility is not a direct result of activity, and point to a role for ubiquitous factors or processes in establishing chromatin accessibility at promoters in the early embryo. However, in concordance with similar works, we find that at enhancers active exclusively in one half of the embryo, we observe a significant skew towards greater accessibility in the region of their activity, highlighting the role of patterning factors such as Bicoid in this process.