PLOS Genetics

Development requires the complex coordination of gene regulatory networks that must remain robust in the face of variable environmental cues. In Caenorhabditis elegans, the nuclear hormone receptor DAF-12 integrates metabolic cues and hormonal signals to control important life history decisions, including development, reproduction, and the rate of aging. Here, we tested the involvement of DAF-12 germline-to-soma signaling in two transgenerational longevity mutants, wdr-5 and jhdm-1. We have previously shown that both mutant populations gradually accumulate repressive H3K9me2 over multiple generations, which is necessary and sufficient for their lifespan extension. We find that daf-12 activity was required for the epigenetic establishment of longevity in both mutant populations, but was only necessary for maintaining longevity in a wdr-5 mutant background. Because DAF-12 also functions as a key regulator of dauer diapause, an alternative developmental stage triggered by environmental stress, we also tested the genetic relationship at earlier points in development. Surprisingly, mutations in either wdr-5 or jhdm-1 rescued the dauer defect of daf-12 mutants, and we found a synergistic effect on unchallenged larval development in wdr-5; daf-12 double mutants. These differing epistatic relationships indicate that, although the acquisition of longevity in both wdr-5 and jhdm-1 mutant populations shares a common mechanism, the impacts on somatic phenotypes (including lifespan extension) proceed via distinct pathways. Together, these results show how heritable chromatin states can co-opt existing developmental programs to influence key developmental decisions. ARTICLE SUMMARYHow do early experiences influence development and aging? In this study, we explore this question by testing the genetic interaction between the DAF-12 signaling pathway and heritable chromatin landscapes. Previously, we showed that two C. elegans mutants can accumulate heterochromatin over multiple generations to acquire longevity. We find that DAF-12 is required to establish this epigenetic trait but is not necessary to maintain it. We also find that chromatin landscapes bypass DAF-12s role earlier in development, including during the decision to enter dauer diapause. Overall, this study shows how chromatin states co-opt existing developmental programs to influence key life history decisions.

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.

During meiosis, homologous chromosomes exchange segments in a process termed crossover recombination. Crossovers are non-randomly distributed along chromosomes, and in many eukaryotes, including plants, meiotic chromosome architecture and chromatin states control recombination landscapes. Whether these two components genetically interact has remained underexplored. To address this question, we combined Arabidopsis thaliana, hereinafter, Arabidopsis, mutations that disrupt meiotic chromosome architecture by depleting the meiotic chromosome axis (asy1/+) or synaptonemal complex (zyp1) with mutations in the DNA methyltransferases MET1 and CMT3 (met1/+ and cmt3), which lead to a loss of cytosine DNA methylation, the hallmark of heterochromatin, in the CG and CHG contexts, respectively. We quantified crossovers in telomere- and centromere-proximal chromosome intervals using fluorescent seed-based reporters and found that DNA methylation and meiotic chromosome architecture proteins can have distinct or cooperative roles in crossover control depending on the chromosome interval and DNA methylation context. We demonstrate that axis and synaptonemal complex act together with CG DNA methylation to control crossovers, while CHG DNA hypomethylation cannot fully restore a loss of centromere-proximal recombination caused by the depletion of ASY1 or ZYP1. Remarkably, increasing ASY1 dosage promotes crossovers within the pericentromere, representing a new non-epigenetic route to upregulate pericentromeric recombination. Author summaryMeiotic crossovers reshuffle genetic variation and are essential for evolution and crop breeding. However, crossovers occur unevenly along chromosomes, limiting genetic exchange in pericentromeric regions. Here, we investigate the genetic interactions between cytosine DNA methylation and meiotic chromosome architecture and show that, although heterochromatin depletion can permit pericentromeric crossovers, the structural integrity of the meiotic chromosome axis and the synaptonemal complex are essential to drive recombination. Remarkably, modulating the dosage of a chromosome axis protein provides a non-epigenetic strategy to increase pericentromeric crossovers, revealing new opportunities to reshape recombination landscapes in model and crop plants.

Cohesin is a large multisubunit protein complex that plays essential roles in cell proliferation, genome organisation, and gene regulation in metazoans. Germline mutations in cohesin subunits or regulators cause a group of human developmental disorders collectively known as cohesinopathies. Increasing evidence indicates that individual cohesin subunits can confer distinct molecular functions to the complex; for example, STAG1 and STAG2 have both overlapping and non-overlapping roles in genome organisation. The zebrafish tailbud provides an excellent developmental model for investigating the coordination of cell proliferation and differentiation, processes in which cohesin has crucial functions. We previously demonstrated that loss of Stag2 disrupts Wnt signalling and mesoderm patterning in the zebrafish tailbud. Here, we show that, unlike mammals, zebrafish can tolerate complete loss of Stag1 from embryogenesis through to adulthood. In contrast to Stag2 deficiency, loss of Stag1 impairs cell cycle progression, activates p53 signalling, and induces a metabolic shift towards catabolism. BMP signalling is reduced in Stag1-deficient embryos and is accompanied by expansion of BMP antagonist chordin expression. Stag1 loss also alters chromatin accessibility at the chordin locus and affects accessibility at chromatin domain boundaries. We propose that modulation of growth and signalling pathways compensates for the absence of Stag1, allowing embryonic development to proceed correctly. Together, these findings reveal distinct contributions of Stag1 and Stag2 to cell-cycle regulation, chromatin architecture, and developmental signalling during vertebrate embryogenesis.

Many neurological diseases are caused by mutations in broadly-expressed genes, but the basis for their neuron-specific manifestation is unclear. In Spinal Muscular Atrophy (SMA), loss of the ubiquitously-expressed spliceosome assembly factor SMN1 causes selective degeneration of motor neurons, leading to progressive neuromuscular decline. We explored the mechanisms of this cell-specific vulnerability using SMA models in the nematode C. elegans, which likewise exhibit progressive neuromuscular defects upon loss of smn-1. Surprisingly, our results show that the intestine - not neurons or muscle - is the selectively-vulnerable tissue causing smn-1 phenotypes. RNA-Seq reveals that loss of intestinal smn-1 causes specific global splicing defects, accompanied by robust transcriptional activation of the Intracellular Pathogen Response (IPR), a stress pathway enriched for ubiquitin-proteostasis genes. Consistent with this, smn-1 mutants exhibit elevated levels of proteasome activity. Pharmacological proteasome inhibition rescues many of the smn-1 mutant defects, as does deletion of specific components of the IPR pathway. These results reveal how the ubiquitously-expressed SMN-1 protein is required in a single tissue to avoid degenerative defects caused by hyperactive proteasome activity, contributing to our understanding of how mutations in ubiquitously-expressed genes can cause highly cell-specific pathologies. SIGNIFICANCE STATEMENTMany ubiquitously expressed genes cause highly selective neurodegenerative diseases, such as Huntingtons disease and Amyotrophic Lateral Sclerosis. The basis for this cell-specific vulnerability remains unclear. We address this question for smn-1 in C. elegans. We show that smn-1 is indeed required in a cell-specific manner, but unexpectedly not in neurons, but rather in the intestine. Both survival defects and behavioral phenotypes originate from intestinal loss of smn-1. We show that these defects are caused by hyperactive protein degradation and immune responses, and that mutant defects can be resolved by reducing these proteostasis and immune pathways using genetics or pharmacology. These results shed light on how a single tissue/cell can dictate the effects of a systemic genetic disease.

With the rise of antimicrobial resistance, anti-virulence therapeutics are a viable alternative to circumvent resistance pressures. Hypothetical genes and proteins are an under-studied source of potential virulence factor targets. We performed bioinformatic analyses to identify conserved hypothetical genes enriched in pathogenic Pseudomonas aeruginosa but not in non-pathogenic strains. This analysis identified an atypical BlaIR system, which we named pvmSR, that regulated P. aeruginosa virulence in a Caenorhabditis elegans infection model. This is in contrast with the typical BlaIR system from Staphylococcus aureus, which regulates resistance to {beta}-lac-tam antibiotics. The{Delta} pvmSR mutant showed reduced virulence in a C. elegans slow-killing assay. To understand how PvmSR regulated virulence in vivo, we performed dual RNA-seq to analyze transcriptomic changes in both C. elegans and P. aeruginosa. We found that C. elegans responded to P. aeruginosa {Delta}pvmSR infection by decreasing expression of lysosome and phagocytosis pathways. In P. aeruginosa {Delta}pvmSR, we observed decreased gene expression of several known virulence factors including the hydrogen cyanide synthase, hcnC, and heparinase, hepP. Additionally, we observed dysregulation in genes important for quorum sensing and biofilm formation. Collectively, our findings indicated that PvmSR contributed to virulence regulation and may serve as a potential anti-virulence target.

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13BV358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated two independent Arl13bV358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13BV358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of GPR161 and TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently-derived, mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses. Summary for ReviewersThis study examined how excluding the protein ARL13B from primary cilia affects kidney epithelial cells. The researchers created two independent cell lines carrying a modified form of ARL13B,along with matched rescue cell lines. The findings show that ciliary ARL13B helps maintain normal ciliary composition. By comparing the cell lines, the researchers identified a high-confidence set of genes associated with loss of ciliary ARL13B. By highlighting the importance of using independent gene-edited clones and rescue-based controls, these results advance understanding of how cilia regulate kidney cell function and provide guidance for designing robust transcriptomic analyses.

The protein complex cohesin plays critical roles in genomic stability by tethering together sister chromatids at their pericentric regions and along their arms from S phase until anaphase. Cohesin-mediated pericentric cohesion prevents aneuploidy by ensuring bipolar attachment of sister kinetochores. Arm cohesion prevents loss of heterozygosity by biasing DNA repair via recombination between sister chromatids rather than between homologs. Here, we investigate in yeast whether cohesin also enhances genomic stability by suppressing unequal sister chromatid exchange (USCE) between repetitive sequences. In wild-type cells, the USCE rate between repeats 4kb apart (proximal) was 15X higher than repeats 68kb apart (distal). The USCE between distal repeats but not proximal repeats increased 4 to 7-fold in mutants with altered cohesin subunits or auxiliary factors. The level of increased distal USCE corresponded with reduced arm cohesion, reduced density of cohesion arm sites, and higher sister loci mobility. Our results suggest that high density of arm cohesion sites confines repair of DNA damage to local sequences. When the density of cohesion sites decreases, sister chromatid sequences are less confined, thereby enhancing distal repeat interactions and USCE. Another set of mutations disrupted both DNA replication and cohesin loading at the replication fork during S phase. Remarkably, distal USCE in these mutants increased approximately 100-fold and was 6-fold more likely than proximal USCE. This preferential hyperdistal USCE can be explained by an aberrant sister-chromatid structure that is normally prevented by proper coupling of cohesin function and replication.

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.

Butterfly wings present a tremendous gallery of colorful patterns, offering a unique opportunity to study how developmental pattern formation processes evolve. We still do not understand the genetic basis of several key aspects of wing pattern development, however. Three paralogous POU domain transcription factors nubbin, ventral veinless (vvl), and pdm3 are all known wing development genes in Drosophila melanogaster. Here we combine gene expression and knockout approaches to show that each of these genes plays multiple novel wing patterning roles in the common buckeye butterfly, Junonia coenia. We found that nubbin controls eyespot pattern determination via a non-cell autonomous repressor-like effect originating at the wing veins, such that nubbin knockouts have larger eyespots. nubbin also regulates pigment identity and scale morphology across the wings. We also found that vvl regulates pigment identity of the discal bands and ventral hindwing. Last, we found that pdm3 is required for determining the outer rings of eyespot patterns, where it is co-expressed with spalt and the lncRNA ivory. pdm3 is also necessary for determining wing margin stripes, where it is again co-expressed with spalt, leading us to propose that the eyespot and wing margin gene regulatory networks could be homologous. Finally, pdm3 affects pigmentation of the ventral hindwing, phenocopying the seasonally-plastic color switch in J. coenia. Together, our work shows that POU domain transcription factors play diverse roles in butterfly wing pattern development and highlights nubbin as one of the first genes implicated in the repressive function of wing veins in color pattern determination. Highlights- Gene expression and knockouts reveal three POU factors regulate butterfly wing color pattern - nubbin regulates eyespot development, likely via a repressor from the wing veins - nubbin controls scale color and morphology across the wing - pdm3 coordinates eyespot development and is co-expressed with spalt and ivory - Expression of genes in the eyespot and wing margin suggests network homology

Across species, sleep amount and timing are tightly linked to the nutritional environment. While early life sleep and sleep in mature organisms are both dramatically influenced by reductions in the dietary environment, the mechanisms linking nutritional cues to conserved sleep-regulatory circuitry are not well understood. Using both early 3rd instar (L3) Drosophila larvae and adults, we examined the plasticity of sleep responses under shifting nutrient environments across the lifespan. We find that L3 larvae and adults exhibit changes in sleep duration in low sugar environments with L3 showing a loss of sleep-wake rhythms that can be rescued with additional nutrients. We show that larval and adult sleep plasticity is regulated by CCHamide-1 signaling between DN1a and Dh44 neurons and glucose metabolic genes in Dh44 neurons. Additionally, our data indicate that sleep plasticity is not dependent on anatomical and functional connectivity between clock-arousal circuitry, suggesting that peptidergic signaling alone is sufficient for diet-dependent sleep regulation. Finally, we demonstrate that Dh44 neurons in both L3 larvae and adults adjust mRNA levels of CCHamide-1 receptor (CCHa1-R) in response to changes in dietary sugar. Together, our findings suggest that organisms utilize conserved molecular signaling pathways across the lifespan to dynamically regulate their sleep in a changing environment.

Neuronal ion channels have well-established effects on synaptic plasticity, in many cases by influencing pathways that depend on membrane excitability. Here we find that a C. elegans two-pore domain potassium (K2P) channel, TWK-40, regulates presynaptic organisation through a membrane potential-independent mechanism. Instead, this mechanism depends on TWK-40's effects on intracellular potassium levels. Loss-of-function mutations in TWK-40 lead to excessive presynaptic protein accumulation, while gain-of-function mutations lead to depleted presynaptic components and cause synaptic transmission deficits. These abnormalities are phenocopied by transporter mutations that mimic TWK-40's effects on intracellular potassium concentration, but not by sodium channel mutations that mimic its effects on membrane excitability. This indicates that cytoplasmic potassium promotes presynaptic assembly. This process depends on the PYK-1 pyruvate kinase, a potassium-sensitive enzyme, and three transcription factors. These findings establish a new pathway linking neuronal potassium homeostasis to the control of presynaptic organisation and synaptic function.

The DUP240 gene family in Saccharomyces cerevisiae encodes ten proteins containing two transmembrane domains (TMDs). Despite decades of interest driven by their high sequence similarity, little functional information exists regarding whether Dup240 family members share redundant or distinct roles. In this study, we combined computational modelling, subcellular localisation, and functional assays across the family to identify shared and unique features. Computational modelling revealed that Ktd1 possesses a unique structural element adjacent to its TMD region. Out of six successfully localised family members, Ktd1 was the only protein predominantly targeted to the vacuolar membrane and the sole Dup240 required for defence against the K28 killer toxin. Computational predictions further indicated that Ktd1 undergoes extensive post-translational regulation, containing multiple validated phosphorylation sites. Screening potential regulatory kinases and phosphatases identified several enzymes required for K28 defence, which were independently validated using liquid-based toxin sensitivity assays. A multicopy suppressor screen demonstrated that KTD1 overexpression rescued K28 sensitivity across most enzyme mutant backgrounds, confirming Ktd1 acts downstream or in parallel to many factors. However, the phosphatase Sit4 and the kinase Hog1 scored as most likely co-factors in Ktd1 mediated defence. Live-cell fluorescence imaging of these two enzymes revealed no dramatic spatial re-localisation during K28 exposure, suggesting that phospho-dependent regulation of Ktd1-mediated defence may occur through transient signalling events. Together, these findings identify Ktd1 as the central effector of the Dup240 family in toxin defence and provide a mechanistic framework for understanding Dup240 regulation.

Environmental stress during early development can have lasting effects on reproduction and developmental plasticity in Caenorhabditis elegans. Here, we compared the consequences of two dauer-inducing stressors, high temperature and crowding, on fertility, dauer formation, and intergenerational gene expression. Entry into the dauer stage protected animals from stress-induced sterility, with high-temperatureinduced diapause (HID) providing strong preservation of reproductive capacity. Remarkably, the progeny of temperature-induced post-dauers (PD-temp) displayed a twofold increase in dauer formation upon re-exposure to heat, revealing a transient intergenerational enhancement of HID. This effect was stimulus-specific, as parental heat exposure suppressed pheromone-induced dauer formation in progeny, while parental pheromone exposure did not enhance HID. This increased dauer propensity was reset after a single stress-free generation. RNA-seq across three generations identified a transient F1-specific gene expression signature associated with enhanced dauer formation upon re-exposure to heat. Functional analyses showed that snpc-1.3, F49F1.7, and Y69A2AR.12 promote HID. In parallel, vit-3 expression was selectively reduced in F1 progeny of PD-temp animals, and vit-3 mutants exhibited increased dauer formation at 27{degrees}C, suggesting that vit-3 normally restrains HID. Consistent with previous work from our group implicating RNAi pathways in environmentally induced diapause and inherited stress responses, we find that endogenous RNAi pathways also modulate HID across generations. Multiple RNAi pathway components contributed to HID, while the nuclear RNAi factor nrde-2 was specifically required for the intergenerational increase in dauer formation. Tissue-specific rescue experiments further suggest that coordinated RNAi activity across tissues contributes differently to parental HID and progeny responses. Together, these findings identify HID as a distinct stress-induced developmental program that transiently modifies progeny responses to recurring thermal stress while preserving reproductive fitness. Our results further indicate that the physiological and intergenerational consequences of dauer entry depend on the environmental cue that induces diapause.

Homing genetic elements are selfish elements that insert themselves into a specific site in a host gene without disrupting its function. They spread through the population because the element codes for an endonuclease that cleaves alleles of the host gene that do not contain the element, leading to DNA repair by gene conversion that increases the elements frequency. Most homing genetic elements in eukaryotes are either self-splicing introns or inteins but we recently discovered a third category, called WHO elements, in the budding yeast genus Torulaspora. WHO elements code for endonuclease proteins with LAGLIDADG motifs and a zinc finger domain, and are related to the mating-type switching endonuclease HO. Their host gene is the aldolase gene FBA1, which is essential. Clusters of up to 9 diverse WHO endonuclease genes are found downstream of FBA1 in different isolates of Torulaspora. Here, we show that there is a genetic conflict between WHO endonucleases and their target site in FBA1. Different alleles of FBA1 vary in their sensitivity or resistance to cleavage by individual WHO endonucleases. We show that a WHO endonuclease recognizes a 28-bp sequence in FBA1 and does not tolerate much sequence variation, but also that this region of FBA1 has experienced positive selection for sequence diversification to evade cleavage. WHO endonucleases and their target site in FBA1 are therefore engaged in an arms race in which each WHO element is under selection to home into other elements, while avoiding being homed into. Significance StatementWHO elements are a recently discovered type of homing genetic element in yeasts, targeting the aldolase gene FBA1. Rather than disrupting FBA1 when they integrate, WHO elements instead replace the 3 half of the gene with an alternative FBA1 3 half. Each WHO element consists of an endonuclease gene and a version of the 3 half of FBA1, and there is high sequence diversity in both genes. We show that there is an evolutionary arms race between WHO endonucleases and their target site in FBA1, which has resulted in rapid evolution of both genes and the formation of clusters of WHO elements at the FBA1 locus.

Antigenic variation in Trypanosoma brucei relies on strict monoallelic expression of variant surface glycoprotein (VSG) genes from a single telomeric expression site (ES), a process sustained by the extranucleolar RNA polymerase I (Pol I) transcriptional body known as the expression site body (ESB). Although the ESB is essential for VSG expression, the mechanisms governing its assembly and maintenance remain poorly understood. Here, we identify SUMOylation as a central regulator of ESB organization and demonstrate that the balance between SUMO conjugation and deconjugation determines the transcriptional state of VSG expression sites. Ectopic expression of the SUMO protease TbSENP disrupted the highly SUMOylated nuclear focus associated with the active-ES, displaced Pol I from its extranucleolar compartment, and markedly increased VSG in situ switching frequency, indicating that continuous SUMOylation is required to preserve ESB integrity. Conversely, targeted recruitment of the SUMO-conjugating enzyme TbUBC9 to a silent ES locally restored SUMOylation, induced de novo formation of an extranucleolar Pol I compartment, activated transcription of the corresponding telomeric VSG gene, and generated stable antigenic switchers expressing the new surface coat. Local SUMOylation preceded Pol I redistribution, supporting a model in which SUMO-dependent interactions nucleate assembly of a transcriptionally competent ESB. Together, our findings identify SUMOylation as both a structural and regulatory determinant of nuclear organization in T. brucei and suggest that dynamic SUMO homeostasis governs the assembly, maintenance, and remodeling of this specialized transcriptional body. Significance StatementAntigenic variation in Trypanosoma brucei depends on the monoallelic expression of Variant Surface Glycoprotein (VSG) genes from a specialized RNA polymerase I transcriptional compartment known as the Expression Site Body (ESB). However, the molecular signals that govern transitions between active and silent expression sites have remained unknown. We show that SUMOylation acts as a reversible molecular switch: disruption of SUMO homeostasis dismantles ESB organization and promotes VSG switching, whereas localized SUMOylation is sufficient to nucleate a functional transcriptional compartment and activate a silent VSG expression site. Our findings establish SUMOylation as a central regulator of nuclear architecture and antigenic variation.

Heterozygous pathogenic variants in CSNK2A1 or CSNK2B encoding the Casein Kinase 2 (CK2) protein complex, lead to pediatric neurodevelopmental disorders, Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS). OCNDS and POBINDS are characterized by a range of symptoms, including developmental delay, intellectual disability, facial dysmorphism, and seizures. Despite over 250 reported cases of OCNDS and POBINDS, we do not fully understand how specific alterations in CK2 relate to the heterogeneity observed in patients. To investigate this, we used the fruit fly, Drosophila melanogaster, as a model system. To assess variant impact, we co-expressed human CSNK2A1 and CSNK2B reference or disease-causing variants in flies. In parallel, we determined the role of Drosophila CkII in the developing and mature nervous system, specifically in neurons and glia. We found that 12/13 variants tested act as full or partial loss-of-function with one CSNK2A1 variant showing gain-of-function. Phospho-proteomic studies in neurons revealed separate signatures for loss- and gain-of-function variants. We found that neuronal and glial CkII is critical for organismal development. Reduction of neuronal CkII in the adult nervous system causes motor and seizure-like phenotypes. Finally, given the known role of CK2 in potentiating Wnt/{beta}-catenin signalling, we show that Wnt agonists partially rescue phenotypes associated with adult-specific neuronal reduction of CkII. This work generates Drosophila models of CSNK2A1 and CSNK2B expression to functionally assess variant impact, as well as an adult-specific neuronal loss-of-function model for drug screening and mechanistic studies.

The QxxR motif is evolutionarily conserved within DEAD-box RNA helicases, including Drosophila Me31B and human DDX6, which post-transcriptionally regulate gene expression during animal development. A pathogenic H372R substitution (QxHR to QxRR) in the QxxR motif of human DDX6 has been associated with various developmental defects, but how this motif contributes to DDX6-family protein function remains unclear. Here, we used Drosophila Me31B as an in vivo model to investigate the QxxR motifs developmental role. We generated a Drosophila strain carrying the corresponding H333R missense mutation in Me31B and characterized its effects on female fertility, embryonic viability, germline development, and Me31B-associated molecular pathways. The me31BH333R mutation reduced female fertility in a gene dose-dependent manner, with homozygous mutant females being sterile. Embryos from the mutant females also exhibited primordial germ cell defects. Despite these developmental phenotypes, the me31BH333R mutation did not significantly alter Me31B protein abundance, global ovarian transcriptome or proteome profiles, or representative germ plasm mRNA and protein localization. In contrast, bait-normalized IP-MS analysis revealed altered enrichment of selected Me31B-associated proteins, including increased association of known Me31B interactors Trailer hitch (Tral) and Ypsilon Schachtel (Yps). These findings establish Me31BH333R as an in vivo model for investigating the conserved QxxR motif and suggest that disruption of this motif compromises development not through broad changes in gene expression, but potentially through altered composition or regulation of Me31B-containing ribonucleoprotein complexes.

The pairwise alignment of homologous chromosomes within the synaptonemal complex (SC) is important for meiotic crossover recombination and fertility. However, chromosomes do not need sequence homology to synapse, with meiotic recombination defects often leading to synapsis of non-homologous chromosome segments. Here we show that such heterologous synapsis reflects a distinctly regulated mode of meiotic chromosome synapsis that also happens during the early stages of wild-type yeast meiosis and occurs in parallel to the well-known synapsis initiation at crossover-designated sites. Heterologous synapsis initiates along chromosome arms after double-strand break resection and is accompanied by canonical markers of crossover repair, but does not need recombinase-dependent strand invasion. Instead, it requires the DNA-damage sensor kinase ATR/Mec1, which phosphorylates of a specific amino acid in Zip1, the major transverse filament protein of the SC. Phospho-mimetic mutants in ZIP1 rescue the synapsis defect of mec1 mutants and also partially restore gamete viability, indicating that this particular MEC1 function is important for the faithful completion of meiosis. Importantly, crossover repair quickly rectifies heterologous synapsis, allowing successful completion of meiosis even when most of the genome initially synapses independently of homology. Our data identify meiotic chromosome synapsis as a dynamic and reversible process that becomes optimized as result of recombination-dependent chromosome pairing.

Mutation rate varies throughout an organisms genome and correlates with many factors, including primary sequence, DNA methylation, gene content, chromatin accessibility, replication timing, and more. Prior work has shown that DNA repair pathways contribute to this variation by repairing certain regions more efficiently than others, sometimes through interactions with gene-associated histone modifications. However, little is known about the relative importance of different DNA repair pathways in shaping intragenomic variability in mutation rate, nor the mutational consequences of perturbing chromatin state. Here, we quantify somatic mutation rate in several DNA repair and chromatin-related Arabidopsis mutants using nanorate sequencing. We find that NER and MMR prevent a smaller fraction of mutations in transposable elements (TEs) compared to other regions of the genome, indicating these pathways are less efficient in heterochromatin. MMR appears to be more efficient in accessible chromatin regions, as its loss nearly abolishes the reduced mutation rate there. TC-NER is the only pathway with greater efficiency in genes than in non-genic non-TE regions, suggesting only TC-NER specifically targets genes. We assay six mutants for histone modifications/variants, but only one (h2a.w.7) displays an altered mutation rate. Instead, mutation rate is elevated in the chromatin remodeler mutant ddm1 and two RNA-directed DNA methylation (RdDM) mutants. The RdDM mutants have a doubled overall mutation rate, but this increase is not localized to RdDM target regions, implicating a transcriptional change, genomic instability, or a secondary function in DNA repair.