PLoS Genetics最新文献

Diversity-Generating Retroelements (DGRs) are specialized genetic systems typically harnessed in nature to evolve new molecular recognition. This mechanism, known as mutagenic retrohoming, relies on an error-prone reverse transcriptase (bRT) that introduces errors at template adenines, followed by the incorporation of the resulting mutagenized complementary DNA (cDNA) into a homologous target gene. Although widely distributed, DGRs are conspicuously absent from key bacterial models, limiting our understanding of their functionality in these hosts and their potential as engineering tools. Here, we demonstrate the 'plug-and-play' nature of the Bordetella phage BPP-1 DGR by successfully reconstituting the mutagenic retrohoming mechanism in Escherichia coli. Using high-throughput tools available in this tractable bacterium, we identified key regulatory factors that allowed us to enhance DGR efficiency over 1000-fold. Systematic analysis defines how sequence context governs bRT's fidelity, uncovering a distinct error profile for the AAC motifs prevalent in natural DGR templates. This intrinsic bias prioritizes the sampling of residues essential for antigen recognition, effectively focusing the evolutionary search within the most productive regions of sequence space. Furthermore, a transposon sequencing screen identified the single-stranded DNA exonuclease ExoI as an inhibitor of DGR activity. While removing ExoI enhanced activity by more than ten-fold, we found that its nuclease activity was dispensable for this inhibition, suggesting a non-catalytic mechanism. Finally, a genome-scale survey highlighted enhanced DGR efficiency at targets located near the replication origin and oriented outwardly from it. This bias is clearly linked to replication directionality, suggesting that certain aspects of DNA replication cycles promote mutagenic retrohoming. Collectively, our work reveals previously unappreciated mechanistic features of DGRs and establishes this reconstituted system as a powerful platform for targeted gene diversification and clarifying the molecular mechanism of mutagenic retrohoming.

Immune responses in tissues display complex spatial patterns of gene expression that are linked to disease outcomes. However, the processes that generate these patterns-including the relative roles of noisy gene expression dynamics, microbial transport, and tissue anatomy-are poorly understood. As a tractable model of spatial immune responses, we investigated heterogeneous expression of antimicrobial peptides in the larval fly fat body, an organ functionally analogous to the liver. To quantify single-cell antimicrobial peptide expression dynamics in the fat body, we developed a protocol for light sheet fluorescence microscopy of whole, live larvae. Using this approach, we discovered that individual fat body cells express antimicrobial peptides at approximately constant rates following infection, but that the average rate varies along the anterior-posterior axis of the fat body, with rapid expression in the anterior and posterior lobes. Overexpression of immune signaling components and analysis of spatial transcriptomes revealed that these tissue microenvironments are predefined independently of infection, with the rate-limiting step of antimicrobial peptide induction downstream of peptidoglycan sensing. The locations of these microevironments correlate with heartbeat-dependent fluid flow in a manner resembling the strategic positioning of immune cells in the liver, gut, and lymph nodes of mammals. We speculate that this spatial compartmentalization helps the fat body efficiently perform its diverse metabolic, enzymatic, and immunological functions.

Retinal detachment (RD) is a sight-threatening emergency requiring urgent intervention to prevent permanent vision loss. While both environmental and genetic risk factors contribute to RD, its complete genetic architecture remains unknown. Here, we performed the largest whole genome sequencing-based case-control study in RD to date, including data from 7,276 RD cases and 236,741 controls in the UK Biobank. Through variant- and gene-level association analyses, we identified VSX2 as a genetic determinant of RD risk while confirming established associations including FAT3, RDH5, and COL2A1. Gene-level collapsing analysis revealed that rare heterozygous missense variants in VSX2 confer a 2.8-fold increased risk of RD (p = 2.4x10-10; odds ratio (OR) = 2.8; 95% confidence interval (CI): [2.1, 3.7]). One missense variant in this gene, p.Glu218Asp, demonstrated a particularly strong effect size (p = 9.3x10-10; OR = 5.9; 95% CI: [3.7, 9.4]). Replication analyses in two additional cohorts, totaling 1,331 cases and 52,355 controls strengthened both the gene- and variant-level associations even further (p = 1.4x10-10 and 1.1x10-11, respectively). Other contributory heterozygous variants included previously reported pathogenic homozygous variants for anophthalmia and microphthalmia. These findings thus reveal a previously unknown gene dosage curve for VSX2, where homozygous mutations cause severe developmental eye disorders and heterozygous mutations cause adult-onset retinal detachment. Extending this observation, we found a significant enrichment for other known recessive Mendelian eye disease genes among nominally significant (p < 0.05) genes associated with RD in the collapsing analysis. This work provides a compelling example of how heterozygous variants in recessive disease genes can be associated with less severe clinical phenotypes.

Long term laboratory-based evolution experiments are a powerful tool that are increasingly being used to study fundamental aspects of evolution and to identify genes that contribute to overall fitness under different conditions. However, even with automation, the time that they take to execute limits the extent to which evolution experiments can be used as part of a high throughput approach to understand the links between genotype and phenotype. Mutations that lead to genetic loss of function (LoF) are frequently selected for in evolution experiments. Thus, in principle these experiments could be done more rapidly by starting not with clonal isolates but with dense transposon libraries that will contain loss of function mutations in all non-essential genes. Here, we test this hypothesis by comparing the results of long term (5 month) evolution experiment, in which E. coli was grown with daily transfers in unbuffered LB starting at pH 4.5, with short term (5 and 10 day) experiments on a high-density transposon library in the same strain and under the same conditions. We show that there is a overlap in the genes and pathways identified using the two methods, as well as identifying other gene of interest whose LoF contributes to fitness. This approach has the potential to complement laboratory-based evolution, enabling rapid, higher throughput, testing of a wide range of parameters that may have an influence on evolutionary trajectories.

Tissues and organs have periods of plasticity that close with age. While period closures can lock in tissue architecture and prevent aberrant cellular interactions, they also limit regenerative capacity. These regenerative periods - a timeframe with regeneration capacity - are defined, but the underlying genetic mechanisms that close specific regenerative periods remains critical knowledge that needs expanding. Here, we established zebrafish larvae as a model to study the genetic basis of regenerative period closure. We demonstrated that laser axotomy of the centrally-projecting axons of dorsal root ganglia (DRG) neurons exhibit a robust regenerative period that is closed by 3 days post fertilization (dpf). The closure of the regenerative period corresponds with the rearrangement of glia that express netrin, introducing the idea that changes in the DCC-mediated signaling axis could be a genetic and molecular basis closing the regenerative period. To test this hypothesis, we manipulated dcc, cAMP, and Rac1 in transgenic animals that label axons and the actin cytoskeleton. Combined with genetic epistasis analysis, we show that altering DCC signaling can re-open the regenerative period, allowing severed axons to regrow into the spinal cord. We show that this increased capacity to reinvade the spinal cord is mediated by growth cone invadopodia. Using calcium reporters and behavioral analysis, we demonstrate that re-opening the regenerative period by manipulating the DCC signaling axis restores the sensory circuit and sensory-specific behaviors. By introducing this genetic basis for regenerative period closure, these results reveal an active suppression process that keeps regenerative periods closed and establishes a new model for future dissection of such periods.

Recent years have witnessed a surge in the development of innovative polygenic score (PGS) methods, driving their extensive application in disease prevention, monitoring, and treatment. However, the accuracy of genetic risk prediction remains moderate for most traits. Currently, most PGSs were built based on the summary statistics from the target trait, while many traits exhibit varied degrees of shared genetic architecture or pleiotropy. Appropriate leveraging of pleiotropy from correlated traits can potentially improve the performance of PGS of the target trait. In this study, we present PleioSDPR, a novel method that jointly models the genetic effects of complex traits and identifies conditions under which leveraging pleiotropy can improve polygenic risk prediction. PleioSDPR models the joint distribution of effect sizes across traits, allowing SNPs to be null for both traits, causal for only one trait, or causal for both traits, and it flexibly captures region-specific genetic correlations and unequal heritability across traits. Through extensive simulations and real data applications, we demonstrate that PleioSDPR improves prediction performance compared with several univariate and multivariate PGS methods, especially when there is no validation dataset. For example, by incorporating information from schizophrenia or leg fat-free mass, PleioSDPR effectively improves the prediction accuracy of bipolar disorder (14.5% accuracy gain) and hip circumference (14.6% accuracy gain), respectively. Moreover, our results indicate that traits with stronger genetic correlations to the target trait, greater heritability, and limited sample overlap contribute more substantially to enhancing prediction accuracy for the target trait. Overall, our study highlights the potential of PleioSDPR to enhance the accuracy of genetic risk prediction by effectively leveraging pleiotropy across traits and diseases. These findings contribute to a broader understanding of polygenic risk prediction and underscore the importance of incorporating pleiotropic information to improve the use of these predictions in disease prevention and treatment strategies.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disease caused by modest alanine expansion at the amino terminus of the nuclear polyadenosine RNA binding protein PABPN1. PABPN1 is expressed ubiquitously and is involved in multiple steps in RNA processing including optimal cleavage and polyadenylation, polyadenylation signal selection, and export of polyadenylated RNAs from the nucleus. Expanded PABPN1 forms aggregates in a subset of muscle nuclei, but PABPN1 levels are paradoxically low in muscle compared to other tissues. Despite several studies in model systems and patient tissues, it remains unclear whether alanine expansion directly impairs PABPN1 function. The molecular mechanisms leading to OPMD pathology are poorly understood. Here we used a proximity labeling approach to better understand the effect of alanine expansion on PABPN1 function in a cell culture model of skeletal muscle. To avoid the confounding factor of overexpression, PABPN1 constructs containing a carboxy-terminal TurboID tag were expressed in skeletal myotubes at near native levels using an inducible promoter. Although non-expanded PABPN1-TurboID was able to complement RNA export and myoblast differentiation defects caused by deficiency of endogenous PABPN1, alanine expanded PABPN1-TurboID was not. Comparative proteomics revealed increased interaction between expanded PABPN1 and RNA splicing and polyadenylation machinery and follow-up studies identified a dominant negative effect of expanded PABPN1 on RNA export in differentiated myotubes. These data indicate that alanine expansion can impair PABPN1 function regardless of the presence of wild type PABPN1 and support a model wherein both loss function and dominant negative effects of expanded PABPN1 contribute to OPMD pathology.

Background: Fibromyalgia, insomnia, depression, and anxiety share common clinical comorbidities, but their underlying genetic architecture and mechanism remain unclear.

Methods: We conducted phenotype-specific Genome-wide association study (GWAS) meta-analyses for fibromyalgia, insomnia, depression, and anxiety, respectively. Genomic structural equation modeling was employed to identify a shared genetic factor (mvFibroPsych). Lead SNPs and associated genes were annotated using Functional Mapping and Annotation (FUMA), followed by gene-set and tissue enrichment analyses. The Latent Causal Variable (LCV) method was utilized to identify modifiable risk factors and phenotypes influenced by mvFibroPsych. Additionally, brain-wide and proteome-wide Mendelian randomization (MR) analyses were applied to explore brain regions and biomarkers associated with mvFibroPsych. Multi-layer molecular quantitative trait locus (QTL) analyses were conducted for mechanistic insights into mvFibroPsych.

Results: Strong genetic correlations were observed among the four phenotypes (rg = 0.55-0.84), with excellent model fit for the common factor [comparative fit index (CFI) = 0.999, standardized root mean square residual (SRMR) = 0.015]. The mvFibroPsych GWAS identified 49 lead SNPs across 43 loci, including 32 novel loci. Gene prioritization revealed 342 protein-coding genes, and pathway analysis indicated enrichment in synaptic function pathway. LCV identified 133 phenotypes causally linked to mvFibroPsych. Brain-wide MR found fractional anisotropy in the splenium of the corpus callosum to be inversely associated with mvFibroPsych. Proteome-wide MR identified five proteins significantly associated with mvFibroPsych, while multi-layer brain QTL analysis prioritized CD40 as a potential target.

Conclusions: This study provides strong evidence for a shared genetic factor underlying fibromyalgia, insomnia, depression, and anxiety, linked to synaptic function, brain structure integrity, and neuroinflammatory pathways.

Copper is an essential micronutrient for all living organisms. Mutations in the copper-importing transporter CTR1/CHCA-1 are associated with a severe copper deficiency disorder in humans, for which no effective cures are currently available. Here, we develop C. elegans as a model for copper deficiency. We show that chca-1 mutant worms fed HT115 bacterial diet exhibited a severe developmental phenotype resulting from copper deficiency, reminiscent of the symptoms observed in human patients. Remarkably, this phenotype can be rescued by switching to OP50 bacterial diet or by supplementing HT115 bacterial diet with glutathione disulfide (GSSG), a metabolite enriched in OP50. Such dietary interventions remodeled the transcriptome of chca-1 mutants towards that of wild-type worms and upregulated the expression of CTR1/CHCA-1-like copper transporters, thereby ameliorating the mutant phenotype. Our findings establish C. elegans as a model for copper deficiency caused by CTR1/CHCA-1, suggesting that dietary interventions may offer a potential therapeutic approach for this severe disease.

Calcium release from intracellular stores influences synaptic response timing and magnitude. Despite the critical role of inositol trisphosphate (IP3)- and ryanodine receptor (RyR)-dependent calcium release in regulating synaptic strength, the upstream signaling mechanisms that govern IP3 receptor or RyR activity remain elusive. Here, we provide evidence that the ArfGAP-containing protein Asap modulates NMJ morphogenesis and synaptic calcium homeostasis by activating IP3-mediated calcium release from the endoplasmic reticulum (ER) via the phospholipase C-beta (PLCβ) signaling pathway. Using CRISPR/Cas9-engineered Asap mutants and genetically encoded calcium sensors, we demonstrate that loss of Asap leads to elevated resting synaptic calcium, resulting in increased evoked amplitude, elevated spontaneous miniature frequency, and reduced synaptic failures under low extracellular calcium conditions. Additional pharmacological and genetic manipulations of calcium regulatory pathways further support the role of increased resting intracellular calcium in driving enhanced neurotransmission in Asap-deficient synapses. Consistent with the role of Asap's ArfGAP domain in NMJ morphogenesis and intracellular calcium regulation, expressing a GDP-locked form of Arf6 (Arf6DN) or knocking down Arf6 in Asap mutants not only rescues Asap-associated synaptic defects but also normalizes synaptic calcium levels. Furthermore, epistatic analysis revealed that attenuation of IP3-signaling components in animals constitutively expressing Arf6CA normalized the NMJ morphological defects and synaptic functions. Together, these findings provide novel insights into the role of Asap-Arf6-PLCβ signaling in IP3-regulated calcium dynamics, sustaining both structural and functional synaptic plasticity.