GENETICS

Conventional LD measures such as r2 perform poorly in the rare common regime, particularly in asymmetric configurations such as nested haplotype structure. Because r2 is symmetric and quadratic, it removes directional structure in two ways: squaring discards the sign, or phase, retained by the signed LD coefficient D, while symmetric normalization hides the asymmetry between the conditional probabilities P(A|B) and P(B|A). Although D recovers the phase, it is locus symmetric and unnormalized; its magnitude is hard to compare across frequency regimes and it does not by itself express which way the asymmetry runs. We therefore analyze the conditional-probability asymmetry {Delta} = P(A|B) - P(B|A), together with r2 and D, as distinct scalar functions on the haplotype simplex under the Fisher information metric. The conditional probabilities P(A|B) and P(B|A) are bounded in [0, 1], directly express carrier-set inclusion, and are more readily visualized than D. Moreover, their difference admits the exact decomposition {Delta} = M + C into a marginal frequency term M and an LD-coupled term C. Prior work has characterized either the mathematical behavior of LD normalizations across allele-frequency space or the Fisher geometry of the haplotype simplex, but not their connection. We bridge this gap by showing that the geometric structure of the simplex explains why LD measures disagree in the rare common regime and why symmetric normalizations such as r2 lose directional information. We show that the fixed-frequency leaf is intrinsically anisotropic, positively curved, and frequency-dependent under the Fisher metric. These geometric predictions are tested empirically , in phased 1000 Genomes data1 and a two locus Wright Fisher model, in a companion paper (Ichikawa, preprint); the present note develops the geometry itself. Keywords: linkage disequilibrium; Fisher information metric; haplotype simplex; rare variant; conditional-probability asymmetry; nested haplotype structure

Many questions in population genetics are concerned with reconstructing evolutionary history through time, such as inferring how population structure has changed throughout the past. Yet, many existing approaches have only an implicit temporal component, using quantities such as allele frequency or haplotype length as rough proxies for age. Recent advances in the inference of Ancestral Recombination Graphs (ARGs) have made it possible to estimate the entire sequence of local genealogies along the genome. These genealogies explicitly encode how samples are related to each other at different time points in the past, enabling the inference of how population structure has changed over time. To this end, recent work has used ARGs to define time-stratified versions of widely-used population genetics summary statistics in an attempt to capture the population structure present within a particular time window. Here, we show that naive approaches result in statistics that cannot be interpreted solely in terms of the population structure present within the time window they are targeting. To address this problem, we introduce a framework of coalescent-based time-stratified statistics, which use coalescence probabilities to partition classical summary statistics into interval-specific contributions. Using coalescent simulations, we demonstrate that these statistics accurately isolate population structure at different temporal depths and avoid spurious signals. Our results highlight the necessity of integrating coalescent theory into ARG-based temporal analyses and provide a principled and practical foundation for studying the dynamics of population structure through time.

Reporters are widely used in Drosophila genetics to visualize gene expression and cell lineages. However, uncharacterized limitations in specific reporter lines can lead to data misinterpretation. Here, we identify a consistent, driver-independent tdTomato signal in the adult proventriculus from the widely used lexAop-tdTomato.nls reporter line. This signal was observed across multiple lexA driver combinations and was directly detectable in lexAop-tdTomato.nls responder-alone adult proventriculi lacking any lexA driver and without antibody staining. In contrast, no comparable native red fluorescence was detected in larval proventriculi under the same no-antibody imaging condition. Mouse and rabbit anti-RFP immunostaining further supported the presence of proventriculus-associated tdTomato/RFP antigen in adult responder-alone animals. In larval responder-alone proventriculi, antibody-amplified staining was antibody-source-dependent: a detectable signal was observed only with rabbit anti-RFP, whereas mouse and rat anti-RFP produced no reliable detectable signal under the same staining condition. A driver-matched comparison using lexAop-RFP.nls did not reproduce the proventricular signal, arguing against detectable ectopic activity of the tested lexA driver in this tissue. However, because lexAop-tdTomato.nls and lexAop-RFP.nls differ in reporter/transgene architecture and possibly genomic insertion context, the underlying cause cannot be assigned specifically to the lexAop sequence. Our findings highlight the necessity of including driver-negative and no-antibody controls when using this reporter line in adult Drosophila proventriculus and gut studies.

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.

Heterogeneous adapting populations, whether in laboratory evolution experiments or global-scale pandemics, experience complex evolutionary dynamics due to the interplay of selection, mutation, and stochasticity. Inference of individual genotypes fitnesses therefore becomes difficult, especially when many lineages are competing and data are noisy. Existing fitness inference methods tend to rely on assumptions on the fitness landscapes maximum order of epistasis, or they require complicated iterative optimization algorithms to converge on fitness estimates. Here, we show that fitness landscapes can be computed from time series data, without any restrictions on epistatic order or iterative optimization, using a simple, closed-form mathematical expression that is easily implemented with standard matrix operations used commonly in linear algebra. We demonstrate successful fitness inference from noisy in silico evolutionary dynamics from four different noisy microscopic processes, including Wright-Fisher, Moran, ProSeD (serial dilution), and barcoded passage simulations. Then, we illustrate the broad applicability of the equation to five experimental time series datasets, including barcoded yeast evolution experiments, murine norovirus-1 serial passage experiments, and SARS-CoV-2 global genomic prevalence data. Our formula successfully infers fitnesses for even for rare genotypes several orders of magnitude less prevalent than top lineages, works with both laboratory evolution and epidemiological data, and can be implemented in most modern scientific programming languages.

Allele frequency (AF) timeseries allow us to directly observe the dynamics of evolution at a genetic level. However, extracting useful inferences from AF timeseries has proved difficult due to the model uncertainties and noisiness inherent in AF change at fine temporal scales. Here we present three new permutation tests --- which do not assume a model of evolutionary change or a parametric statistical model --- to detect AF timeseries features of evolutionary interest. The features identified by these approaches are: 1) any evolutionary change (as opposed to apparent change due to measurement error); 2) directional selection; 3) fluctuating selection with a propensity to change sign (negative autocorrelation). We are not aware of existing tests for features 1 and 3. Feature 2 is commonly tested using standard evolutionary models such as the Wright-Fisher; we show that the permutation approach has comparable statistical power. We apply our new approaches to AF timeseries data from D. melanogaster and D. pulex.

The Saccharomyces cerevisiae Pch2 protein is a conserved meiotic AAA+ ATPase whose activity must be tightly regulated to ensure proper chromosome dynamics during meiotic prophase I. Its function relies on remodeling the HORMA-domain protein Hop1, promoting conformational transitions that are essential for chromosome axis organization, checkpoint signaling, and recombination control. Here, we identify threonine 428 (T428), located within a conserved threonine-glutamine (TQ) putative phosphorylation motif, as a critical regulatory residue of Pch2. We found that, in zip1{Delta} cells, the meiotic recombination checkpoint response is partially or completely abolished in the pch2-T428A and pch2-T428D mutants, respectively. Both mutations alter Pch2 subcellular localization, leading to its increased nuclear accumulation; however, forced nuclear exclusion of Pch2-T428A, but not Pch2-T428D, restores the zip1{Delta} meiotic block, indicating an additional effect of the T428D substitution on checkpoint function beyond subcellular distribution. Analysis in synapsis-proficient strains reveals that this residue also plays a critical role in coordinating Hop1 chromosomal enrichment with Mek1 activation along the synaptonemal complex. In contrast to pch2{Delta} or the ATPase-defective pch2-E399Q mutant, introduction of a negative charge at the 428 position uncouples Hop1 accumulation from its phosphorylation, preventing Mek1 activation despite robust Hop1 association with meiotic chromosomes. These findings support emerging models in which Pch2 regulates Hop1 to control not only its chromosomal abundance, but also the maintenance of sufficient levels of Hop1 in a phosphorylation-competent conformation, thereby ensuring proper checkpoint signaling and faithful meiotic progression.

Restricting the localization of centromere-specific histone H3 variant Cse4 (CENP-A in humans) to centromeric chromatin is essential for chromosome segregation. Mislocalization of overexpressed Cse4/CENP-A to non-centromeric regions contributes to chromosomal instability (CIN) in model organisms and human cells. CIN is an important hallmark of many cancers and hence defining mechanisms that prevent mislocalization of Cse4 is clinically significant. Here we report a role for YRA2 (Yeast RNA Annealing Protein 2) in ubiquitin mediated proteolysis of Cse4 to prevent its mislocalization for chromosomal stability. YRA2 was identified in a genome-wide screen for gene deletions that exhibit synthetic dosage lethality (SDL) upon overexpression of CSE4 (GALCSE4). We determined that yra2{Delta} strains exhibit increased Cse4 stability, enriched Cse4 chromatin association, reduced Cse4 ubiquitination, Cse4 mislocalization, and CIN. Defects in interaction of E3 ubiquitin ligase Psh1 with Cse4 contributes to stability of Cse4 in yra2{Delta} strains. Consistent with these results, overexpression of PSH1 suppresses GALCSE4 SDL in yra2{Delta} strain. We determined that Yra2 mediated proteolysis of Cse4 is independent of its RNA related functions as strain deleted for the C-terminal ChTOP domain of Yra2 with an intact N-terminal RNA binding domain exhibits GALCSE4 SDL and defects in Cse4 proteolysis. Furthermore, poly(A)+ RNA export mutants in YRA1 (yra1-2) and MEX67 (mex67-5), that interact with Yra2, do not exhibit GALCSE4 SDL and defects in RNA export are not observed in yra2{Delta} cells. In summary, we have defined a key role for Yra2 in preventing mislocalization of Cse4 by facilitating its proteolysis to preserve chromosomal stability. Article summaryAccurate segregation of chromosomes during cell division is essential because segregation errors are linked to cancer and developmental disorders. We investigated how cells prevent mislocalization of centromere-specific histone H3 variant Cse4, which is essential for faithful chromosome segregation. We found that the yeast RNA annealing protein Yra2 prevents Cse4 mislocalization by promoting Psh1 mediated ubiquitination and degradation of Cse4. Cells lacking Yra2 showed increased stability of Cse4, enhanced chromatin enrichment with mislocalization to non-centromeric regions and CIN. These defects were suppressed by induction of Psh1. Our findings reveal a novel role for Yra2 in regulating Cse4 levels for chromosomal stability.

In studies of assortative mating, similarity between variables measured in parents is often quantified using correlation. The order of the parents within any given pair can be arbitrary in these applications, but common correlation estimators are not robust to reordering within pairs. These unordered variable pairs are exchangeable, since the joint distributions of both orders are equal, and a given order is biased if the one variable has a lower expectation than the other. In this work, we characterize the effect of order bias on Pearson correlation estimates assuming exchangeable variables, and develop a new unbiased estimator, CorSym, that does not depend on order within each pair. Exchangeable variables have equal marginal distributions for both variables, a property accounted for by CorSym. In contrast, standard correlation estimators assume the two variables have different distributions, so biased orders skew the underlying mean, variance and covariance estimates. We show, through theory and simulations, how order bias often results in upwardly biased Pearson correlation estimates. Simulations confirm CorSym is unbiased, and validate its estimated confidence intervals. Using real admixed trios (parents and a child) from 1000 Genomes, we first demonstrate that the global ancestry of fathers and mothers are consistent with exchangeability, using both Kolmogorov-Smirnov tests and a Binomial test for order bias. However, ANCESTOR, which estimates parental global ancestry from a child's local ancestry, produces significant order biases in its output that result in substantial Pearson biases, which CorSym overcomes. Compared to ancestry proportions calculated directly on the parents, ANCESTOR also overestimates parent ancestry divergence and experiences another estimation artifact. Overall, CorSym solves an important estimation bias likely to be encountered in the study of assortative mating, providing unbiased and deterministic estimates that do not depend on the arbitrary order of the data.

Linkage disequilibrium between two biallelic loci is usually summarized by scalar association measures such as r2 and D'. These measures quantify how visible an allelic association is to a symmetric LD scan, but they do not directly represent the topology of carrier sets: whether the carriers of one variant are contained within, partially overlap with, or are disjoint from the carriers of the other. This distinction is structural. On the haplotype-frequency simplex, carrier-set inclusion corresponds to a boundary face where one haplotype class is absent. In the rare-common regime, a nested rare variant is further constrained by the ceiling r2 [≤] pA/pB, so that complete carrier-set inclusion can remain nearly invisible to r2. Here, as a companion to the Fisher-geometry preprint 1, we examine the empirical and dynamic behavior of this carrier-set topology. In 1000 Genomes Phase 3, across 156,604,320 SNP pairs from the MHC and NEGR1 regions, pairs on the | D' |= 1 boundary span a wide range of r2 and | C |. Within fixed r2 strata, r2 poorly distinguishes nested from non-nested carrier-set configurations, with AUROC values of approximately 0.54-0.62, whereas the boundary-sensitive normalization | D' | separates them much more effectively, with AUROC values of approximately 0.90-0.92. The empirical data also obey the predicted r2 [≤] pA/pB ceiling. We then introduce a temporal axis using a two-locus Wright-Fisher model on the same simplex. Carrier-set topology evolves through three motions relative to the | D' |= 1 boundary: formation or persistence, in which recombination suppression establishes and maintains inclusion without requiring selection; visibility change, in which selection or drift moves r2 along the boundary while preserving the inclusion relation; and breaking, in which a recombination pulse introduces the previously absent haplotype and dissolves inclusion. A fourth mode, specificity erosion, expands the partner carrier set while preserving inclusion, thereby lowering P(A | B) while keeping P(B | A) and | D' | equal to one. This mode shows that asymmetric conditional probabilities are best understood as diagnostic coordinates for carrier-set topology, not as the primary object itself. Together, these results show that topology and visibility are separable axes of LD structure. Conventional r2-based scans and carrier-set topology scans therefore answer complementary, not interchangeable, questions.

Biobank-scale datasets now routinely include hundreds of thousands to millions of individuals, and as sample sizes grow, close relatives become increasingly prevalent. The convention in population genetics has been to remove close relatives prior to inference, effectively treating them as a nuisance parameter. However, the consequences of this practice for demographic inference, and specifically for estimates of recent effective population size (Ne), have not been rigorously evaluated. Here, we benchmark IBDNe and HapNe-IBD, two widely-used methods for inferring recent Ne from identity-by-descent (IBD) segments, under a range of demographic histories and relative sampling schemes. We show that when individuals are randomly ascertained, retaining all relatives produces the least biased Ne estimates; in contrast, removing even second-degree relatives inflates recent Ne and induces oscillatory artifacts that "ripple", leading to biased estimates up to ten generations into the past. We demonstrate that this ripple effect arises because close relatives contribute IBD segments that are assigned by the model to a range of ancestral ages beyond their true TMRCA, meaning their removal creates signal deficits across multiple generations simultaneously. We further show that deliberately oversampling close relatives produces severe downward bias in recent Ne. To support these analyses, we develop an open-source IBD simulation pipeline using msprime that generates realistic IBD segments under arbitrary demographic histories and Wright-Fisher pedigrees. We provide practical guidelines for IBD simulation schemes incorporating pedigrees and argue that, in the biobank era, retaining close relatives is generally the best practice for IBD-based Ne inference.

Population bottlenecks profoundly shape genetic diversity, but distinguishing stochastic drift from selective pressure requires precise estimation and accounting for bottleneck size. While deep-sequencing data enable inference via frameworks like beta-binomial modeling, integrating these estimates directly into selection tests remains a critical challenge. In this study, based on existing computational approaches, we propose a new method that explicitly incorporates bottleneck size estimates into neutrality tests for biological diversity data. Designed for variant frequency data, our framework accounts for sequencing errors and sampling biases to improve the precision and interpretability of selection signature detection. We validate this framework using previously published Streptococcus pneumoniae in vivo experimental data, successfully replicating established fitness results, while uncovering novel genes relevant to infection and pathogenesis. This integrated new model with explicit bottleneck effects narrows down the set of candidate genes under selection and provides a robust, generalizable tool for disentangling drift from selection across a wide range of biological systems.

Gene drive systems enable rapid spread of desired transgenes throughout populations. Advances in CRISPR technology have facilitated the construction of toxin-antidote gene drives, which utilize a CRISPR nuclease as the toxin to disrupt an essential wild-type gene alongside a recoded version of the same gene as the antidote. Because these systems propagate by eliminating wild-type alleles rather than directly copying themselves like homing drives, they typically exhibit introduction thresholds, allowing them to be confined to target populations. Previous work developed the efficient Toxin-Antidote Recessive Embryo (TARE) drive, but its threshold may be too low in challenging confinement scenarios. Here, we constructed a 2-locus TARE drive system. It has underdominance characteristics, yielding a higher introduction threshold, even when drive performance is ideal. It targets the essential but haplosufficient genes hairy and sim using two different drives at different genomic locations, each targeting the gene that the other rescues. Our system involved two linked elements together with rare homology-directed repair-mediated drive conversion, reducing the threshold to compensate for fitness costs. The system showed high efficiency in individual crosses. When released into multigenerational cage populations above the introduction threshold, the drive successfully and rapidly modified the entire population, and when below this threshold, it was eliminated. Our findings indicate that 2-locus TARE drives represent promising tools for effective and strongly confined population modification.

Import of double-stranded RNA (dsRNA) into the germ line can have consequences that last for many generations. However, the role of such transgenerational regulation by extracellular dsRNA is unclear. In the nematode C. elegans, entry of dsRNA into the cytosol requires the transmembrane protein SID-1 and loss of SID-1 for a few generations causes changes in gene expression that can persist for hundreds of generations. Here we report an expanded number of such SID-1-dependent genes (SDGs) and analyze two germline-expressed SDGs: sdg-1 and sdg-2. Deleting sdg-1 reduces brood size in some lineages. An endogenous SDG-1::mCherry fusion protein shows conditional enrichment within nuclei, colocalization with perinuclear germ granules, and colocalization with microtubules. Although animals with SDG-1::mCherry have a normal brood size, they have fewer early progeny with some animals showing defective germline morphology. Deleting the sdg-1 open reading frame eliminates defects in most but not all the animals that express mCherry in a now sdg-1(-) background, suggesting transgenerational consequences of SDG-1::mCherry that persist in some siblings lacking sdg-1. Deleting sdg-2 also reduces brood size in some lineages. An endogenous SDG-2::mCherry fusion protein is constitutively detectable in the cytoplasm and nucleus. The sequence and predicted structure of SDG-2 suggest that it can interact with the Gli-type transcription factor TRA-1, which regulates spermatogenesis. Together, these results suggest that changes in SDG-1 or SDG-2 can impact reproduction. Therefore, the import of extracellular dsRNA or other SID-1 function(s) that regulate SDGs could have evolved to modulate the lingering impacts of ancestral epigenetic changes.

Maternally transmitted Wolbachia often cause cytoplasmic incompatibility (CI), a sperm modification that kills host embryos lacking the endosymbiont. CI produces defects in paternal chromosome replication, condensation, and segregation during the first zygotic cell cycle, but a significant fraction of embryos progress normally through this and subsequent cycles and only exhibit defects at later developmental stages. These results, together with documented CI-induced epigenetic chromatin modifications, suggest heritable chromatin modifications are responsible for the developmentally delayed defects. Here, we conducted a Position-Effect Variegation (PEV) screen in Drosophila melanogaster using In(1)wm4to test for persistent effects on heterochromatin-mediated silencing in adults that survived CI. We show that Wolbachia acts as a variegation suppressor, or Su(var), increasing eye pigment when present in CI-inducing fathers, a reproducible effect observed across several maternal genotypes that differed in CI strength. That is, passage of the In(1)wm4through Wolbachia-infected males limits the spread of heterochromatin into the neighboring euchromatin in the progeny. This effect is consistent with disruption of heterochromatin establishment at the mid-blastula transition, when stochastic spreading of heterochromatin determines whether the displaced white gene is silenced. Surprisingly, maternal Wolbachia did not revert the PEV modification, and in one genotype, Wolbachia increased suppression. Together, our results demonstrate that Wolbachia-mediated chromatin effects persist to adulthood, are not corrected by CifA-dependent rescue, and can be compounded by maternal Wolbachia. These findings establish that rescue is incomplete at the level of heterochromatin-mediated silencing and suggest that CI-specific and constitutive Wolbachia chromatin effects may operate through at least partially independent pathways.

The P-element, a DNA transposon, has independently invaded two Drosophila species, accompanied by rapid evolution of suppression. In the germline, suppression is mediated primarily by maternally expressed piRNAs, a class of regulatory small RNAs associated with PIWI proteins. The offspring of females that lack P-element-specific piRNAs and males that contain P-elements suffer a syndrome of deleterious phenotypes, including sterility, genome rearrangements, gonadal atrophy, and mutations, while the offspring of the reciprocal cross are normal. These effects, collectively termed hybrid dysgenesis, have been investigated primarily in female D. melanogaster. Here, we study hybrid dysgenesis in male D. simulans. Using an attached-X chromosome stock, we generated genetically identical F1 males that differed only in maternal suppression of the P-element. Using targeted sequencing of P-element breakpoints, we show that P-element transposition is elevated in dysgenic males and confirm a preference for insertion near origins of replication. Using transcriptomics, we show that dysgenic males have elevated P-element expression and reduced splicing suppression, with patterns of gene expression suggesting the loss of mature sperm cells. Fertility assays show higher rates of male sterility but otherwise modest effects on fertility. In conjunction with the transcriptomic data, small RNA sequencing confirms that the piRNA pathway functions in testes. Our results suggest that the P-element may spread more readily through males than females, as transposition rates are similar while fertility defects are less severe in males.

Eusocial Hymenoptera present the highest known recombination rates among metazoans, which evolved several times independently among bees, ants and wasps. Several hypotheses have been proposed to explain this observation, including stronger selection for recombination caused by coevolving parasites and pathogens, and strong sexual selection among haploid males due to male-biased sex ratios among reproductive individuals. In this article, we explore the effects of haplodiploidy and differential selection between sexes on the evolution of recombination, by analyzing a three-locus model in which selection for recombination stems from negative epistasis between selected loci. Our analytical predictions are compared with the results of individual-based simulations in which deleterious mutations occur along a linear chromosome. Our results show that, at mutation-selection balance for deleterious alleles, increasing the strength of selection against deleterious alleles (due to the effect of male haploidy and/or sexual selection) tends to reduce selection for recombination. However, an increase in the overall magnitude of negative epistasis (which may also be due to male haploidy and/or sexual selection) combined with the fact that recombination only occurs in females may increase selection for recombination substantially. Our model also shows that, in conditions favoring recombination, increasing recombination in meioses leading to parthenogenetic ovules (and male offspring) may yield stronger benefits than in meioses leading to fertilized ovules (and female offspring).

Insulin/IGF signaling (IIS) inhibits the nuclear localization of the DAF-16/FOXO transcription factor to regulate longevity and stress resistance in C. elegans. In the intestine, IIS promotes DAF-16 localization to endosomes and loss of TBC-2, a RAB-5 GAP, results in increased endomembrane localization of DAF-16 at the expense of nuclear localization, decreased DAF-16 target gene expression, longevity and stress resistance. Here we found that TBC-2 differentially regulates the localization of the IIS-regulated transcription factors PQM-1 and HLH-30/TFEB. Our results suggest a broader role for TBC-2 in negatively regulating IIS and that TBC-2 likely functions at an upstream point in the IIS pathway.

The conversion of transcriptionally silent GGAA microsatellites (GGAASats) into functional enhancers by FET::ETS oncogenic fusions is a hallmark of Ewing sarcoma. However, emerging evidence implicates non-fused, full-length oncogenic ETS transcription factors in activating these repeats in other malignancies. Evaluating the in vivo transcriptional requirements of various human ETS factors in Drosophila, we found that human ETV4 uniquely binds and robustly activates GGAASats in a tissue-specific manner. This activation is strongly inhibited by the human ETS repressor ETV6. Taking advantage of low genetic redundancy in Drosophila, we identified Cabeza (Caz), the single fly FET orthologue, as a necessary cofactor for ETV4-mediated transcription at GGAASats. Conversely, EWS::FLI1-mediated transcriptional activation of GGAASats is entirely independent of endogenous Caz, highlighting the distinct mechanics of covalent tethering versus non-covalent physical complexes. Collectively, our findings provide definitive in vivo evidence that non-fused ETS factors cooperate with endogenous FET proteins to drive transcription from silent GGAA repeats, mechanistically validating this regulatory transformation known to operate as an oncogenic mechanism beyond Ewing sarcoma.

Genotype-by-environment interactions (GxE), or differences in how genotypes perform across varying environments, are a pervasive source of phenotypic variation and underlie differences in local adaptation. Though GxE is well characterized across kingdoms of life, less is known about what causes GxE interactions, particularly at the molecular level. In this study, we use allele-specific gene expression estimates in a maize (Zea mays L.) B73 x Mo17 hybrid to isolate cis-regulatory effects on gene expression for each of the two parental alleles. The hybrid was grown in two environments, and expression differences between the parental alleles were used to characterize allele-by-environment (AxE) interactions and study the influence of gene-proximal sequence variation on transcript abundance AxE. We tested the hypothesis that gene-proximal sequence variation can cause GxE in gene expression by modifying transcription factor binding. Our results show that sequence variation in gene promoter regions has a small but consistent enrichment in genes that show transcriptional AxE. Further, we demonstrate that differential transcription factor binding potential caused by sequence variation is also enriched in AxE genes. Predictive models trained on sequence and transcription factor binding variation show that while these features contain some information about whether a gene will show transcriptional AxE, they alone are not sufficient to reliably distinguish AxE genes. These findings support the hypothesis that gene expression GxE can be caused by sequence variation that modifies transcription factor binding, while also reinforcing the complex and context-specific nature of GxE interactions.