PLoS Genetics最新文献

Most of the disease associated genetic variants identified in genome wide association studies have been mapped to the non-coding regions of the genome. One of the leading mechanisms by which these variants are thought to affect disease susceptibility is by altering transcription factor (TF) binding. Even though inbred mouse strains have been commonly used to investigate polygenic diseases, less is known on how their genetic differences translate to the level of gene regulation and chromatin landscape. Here, we investigated how genetic variation affects chromatin accessibility in the epididymal white adipose tissue (eWAT) of C57BL/6J and 129S1/SvImJ mice, which are commonly used to study diet-induced obesity, fed either chow or high-fat diet. We show that differences in chromatin accessibility are almost exclusively strain-specific and driven by genetic variation. In addition, we integrate ATAC-seq (chromatin accessibility) and H3K27ac ChIP-seq (active regulatory regions) data to show that tissue-specific TF binding sites are commonly found in the active regulatory regions hosting TF motif altering variants in eWAT. Using footprint analysis, we also show that TF occupancy is consistent with TF binding motif scores at the genetically altered loci. In addition, we validate these findings by extending the analysis to ATAC-seq and H3K27ac ChIP-seq data obtained from the liver. We employ RNA-seq to show that differentially expressed genes are co-located with differentially accessible regions hosting genetic variants. Overall, our findings highlight the connection between differential chromatin accessibility and genetic variation across metabolically central tissues of a mouse model for polygenic obesity.

Genetic compensation is a common phenomenon in zebrafish in response to genetic alterations. Differences between genetic and morpholino-mediated zebrafish models of human diseases have led to significant difficulties in phenotypic interpretation and translatability. One form of compensation is the maternal deposit of mRNAs and proteins to the oocyte that supports developmental processes before zygotic genome activation. In this study, we generated a zebrafish model of severe congenital muscular dystrophy (CMD) by targeting protein O-mannose N-Acetylglucosaminyltransferase 2 (pomgnt2), a maternally provided gene that maintains cell-extracellular matrix interactions through glycosylation and leads to congenital muscular dystrophy when mutated. Zygotic knockouts (ZKOs) retain protein function in the first week post fertilization and survive to adulthood, only developing muscle disease later in life. In contrast, maternal-zygotic KOs (MZKOs) generated from ZKO females develop early-onset muscle disease, reduced motor function, neuronal axon guidance deficits, and retinal synapse disruptions recapitulating features of the human presentation. While assessing transcriptional changes linked to disease progression, the availability of embryos obtained from different breeding strategies also allowed for a direct comparison of ZKOs and MZKOs to define the impact of having a KO mother. We found that offspring from a ZKO mother, independently of genotype, show distinct expression patterns from animals obtained from heterozygous breedings. Some of these changes reflect changes in metabolic function, possibly stemming from maternal metabolic disruption. These findings will not only be applicable for other CMD models targeting maternally provided genes, but also provide new insight into modeling disease using maternal-zygotic mutants.

Neuronal injury due to trauma or neurodegeneration is a common feature of aging. The clearance of damaged neurons by glia is thought to be critical for maintenance of proper brain function. Sleep loss has been shown to inhibit the motility and function of glia that clear damaged axons while enhancement of sleep promotes clearance of damaged axons. Despite the potential role of glia in maintenance of brain function and protection against neurodegenerative disease, surprisingly little is known about how sleep loss impacts glial function in aged animals. Axotomy of the Drosophila antennae triggers Wallerian degeneration, where specialized olfactory ensheathing glia engulf damaged neurites. This glial response provides a robust model system to investigate the molecular basis for glial engulfment and neuron-glia communication. Glial engulfment is impaired in aged and sleep-deprived animals, raising the possibility that age-related sleep loss underlies deficits in glial function. To define the relationship between sleep- and age-dependent reductions in glial function, we used two complementary approaches to enhance sleep in aged animals and examined the effects on glial clearance of damaged axons. Both pharmacological and genetic induction of sleep restores clearance of damaged neurons in aged flies. Further analysis revealed that sleep restored post-injury induction of the phagocytic protein Draper to aged flies, fortifying the notion that loss of sleep contributes to reduced glial-mediated debris clearance in aged animals. To identify age-related changes in the transcriptional response to neuronal injury, we used single-nucleus RNA-seq (snRNA-seq) of the central brains from axotomized young and old flies. We identified broad transcriptional changes within the ensheathing glia of young flies, and the loss of transcriptional induction of autophagy-associated genes. We also identify age-dependent loss of transcriptional induction of 18 transcripts encoding for small and large ribosomal protein subunits following injury in old flies, suggesting dysregulation of ribosomal biogenesis contributes to loss of glial function. Together, these findings provide further support for a functional link between sleep loss, aging and Wallerian degeneration.

The mitochondrial genome (mtDNA) encodes essential subunits of the electron transport chain and ATP synthase. Mutations in these genes impair oxidative phosphorylation, compromise mitochondrial ATP production and cellular energy supply, and can cause mitochondrial diseases. These consequences highlight the importance of mtDNA quality control (mtDNA-QC), the process by which cells selectively maintain intact mtDNA to preserve respiratory function. Here, we developed a high-throughput flow cytometry assay for Saccharomyces cerevisiae to track mtDNA segregation in cell populations derived from heteroplasmic zygotes, in which wild-type (WT) mtDNA is fluorescently labeled and mutant mtDNA remains unlabeled. Using this approach, we observe purifying selection against mtDNA lacking subunits of complex III (COB), complex IV (COX2) or the ATP synthase (ATP6), under fermentative conditions that do not require respiratory activity. By integrating cytometric data with growth assays, qPCR-based mtDNA copy-number measurements, and simulations, we find that the decline of mtDNAΔatp6 in populations derived from heteroplasmic zygotes is largely explained by the combination of its reduced mtDNA copy number-biasing zygotes toward higher contributions of intact mtDNA-and the proliferative disadvantage of cells carrying this variant. In contrast, the loss of mtDNAΔcob and mtDNAΔcox2 cannot be explained by growth defects and copy-number asymmetries alone, indicating an additional intracellular selection against these mutant genomes when intact mtDNA is present. In heteroplasmic cells containing both intact and mutant mtDNA, fluorescent reporters revealed local reductions in ATP levels and membrane potential ([Formula: see text]) near mutant genomes, indicating spatial heterogeneity in mitochondrial physiology that reflects local mtDNA quality. Disruption of the respiratory chain by deletion of nuclear-encoded subunits (RIP1, COX4) abolished these physiological gradients and impaired mtDNA-QC, suggesting that local bioenergetic differences are required for selective recognition. Together, our findings support a model in which yeast cells assess local respiratory function as a proxy for mtDNA integrity, enabling intracellular selection for functional mitochondrial genomes.

The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay sarcopenia onset. Identification of therapeutic targets to alleviate the symptoms of aging requires in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish undergoes aging, with hallmarks including mitochondrial dysfunction, telomere shortening, and accumulation of senescent cells. However, zebrafish age slowly, and no specific zebrafish models of accelerated muscle atrophy associated with molecular events of aging are currently available. We have developed a new genetic tool to efficiently accelerate muscle-fiber degeneration and muscle-tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin-1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated neuromuscular atrophy, the atrofish. We demonstrated that Atrogin-1 expression specifically in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunction in both larvae and adult fish. We identified degradation of the myosin light chain as an event occurring prior to muscle-fiber degeneration. Biological processes associated with muscle aging such as proteolysis, inflammation, stress response, extracellular matrix (ECM) remodeling, and apoptosis are upregulated in the atrofish. Surprisingly, we observed a strong correlation between muscle-fiber degeneration and reduced numbers of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while atrofish larvae can recover locomotive functions, adult atrofish have impaired regenerative capacities, as is observed in mammals during muscle aging. In the future, the atrofish could serve as a platform for testing molecules aimed at treating or alleviating the symptoms of muscle aging, thereby opening new therapeutic avenues in the fight against sarcopenia.

We measured genomic responses to active vitamin D, 1α,25-dihydroxyvitamin D (1,25D), in colonic organoids from individuals of African and European ancestry. Given protective effects of 1,25D for gastrointestinal conditions such as colorectal cancer, organoid cultures enabled evaluation of condition-specific responses in relevant target tissue across individuals of diverse ancestries. We found significant alterations in transcriptional and chromatin accessibility responses to 1,25D treatment, including some with ancestry-associated differences, and also elucidated the role of cis-genetic variants on treatment responses. Integration of genomic profiling with genetic mapping found an insertion-deletion variant that explains ancestry-associated differences in 1,25D regulation of POLB, an oxidative DNA repair enzyme involved in colorectal carcinogenesis, which also showed signals of positive natural selection. These findings highlight the importance of including diverse individuals in functional genomics studies to identify potential drivers of population-level differences relevant for clinical outcomes, and to uncover functional mechanisms that may be obscured by ancestry variation.

Male sterility (MS) plays a crucial role in plant reproduction and hybrid breeding as it is associated with pollen viability and release. However, the regulatory mechanisms governing anther dehiscence in peppers remain poorly characterized. Thus, this study identified the pepper C2H2 family transcription factor CaZAT5 and characterized its function. The results indicated that CaZAT5 represses transcriptional activity and is predominantly expressed during pepper flower development. Silencing CaZAT5 in pepper led to early flowering, whereas its overexpression (OE) in tomato delayed flowering. Moreover, CaZAT5 negatively regulated vegetative growth by suppressing CaSOC1 expression, thereby affecting pollen morphology and viability. Histological analyses revealed that the anthers of CaZAT5-OE plants exhibited abnormal mitosis, resulting in both enlarged and shrunken pollen grains. Additionally, CaZAT5 overexpression inhibited anther dehiscence during pollen maturation, affecting pollen release. The consequent reduction in pollen viability and inhibited anther dehiscence decreased fruit set and yield in the plants. Transcriptome (RNA-seq) analysis revealed that CaZAT5 overexpression suppressed the expression of genes involved in cell wall loosening, degradation, and secondary wall thickening in the anthers. DAP-seq, Y1H, Dual-LUC, and EMSA identified potential CaZAT5-regulated genes involved in anther dehiscence, including cell wall degradation genes (CaPG and CaBG4) and the expansin gene CaExpA13. Collectively, these findings suggest that CaZAT5 modulates flowering time, pollen development, and anther dehiscence by regulating the expression of genes related to flowering and cell wall loosening and degradation. These findings contribute to a more comprehensive understanding of the potential role of CaZAT5 in regulating flowering time and male fertility.

The ability of genomic inversions to reduce recombination and generate linkage can have a major impact on genetically based phenotypic variation in populations. However, the increase in linkage associated with inversions can create hurdles for identifying associations between loci linked to inversions and the traits they impact. Therefore, the role of inversions in mediating genetic variation of complex traits remains to be fully understood. This study uses the fruit fly Drosophila melanogaster to investigate the impact of inversions on trait variation. We tested the effects of common inversions among a diverse assemblage of traits including aspects of behavior, morphology, and physiology, and identified that the cosmopolitan inversions In(2L)t and In(3R)Mo are associated with many traits. We compared the ability of different approaches of accounting for relatedness and inversion presence during genome-wide association to identify signals of association with SNPs. We report that commonly used association methods are underpowered within inverted regions, while alternative approaches such as leave-one-chromosome-out improve the ability to identify associations. In all, our research enhances our understanding of inversions as components of trait variation and provides insight into approaches for identifying genomic regions driving these associations.

The doublesex and mab-3 related transcription factor 1 (dmrt1) plays a crucial role in metazoan sexual differentiation. This gene, or its paralogs, independently became triggers for sex determination several times, including in the tetraploid African clawed frog Xenopus laevis. To explore functional evolution of this gene, we generated knockout lines of each of two dmrt1 homeologs in X. laevis and an ortholog in the closely related diploid Western clawed frog X. tropicalis. Our findings evidence sex-specific functional evolution following duplication by allotetraploidization in an ancestor of X. laevis. In females, dmrt1 was essential for fertility and oogenesis in the Xenopus ancestor, but this important function was lost (subfunctionalized) in one X. laevis homeolog (dmrt1.S) after allotetraploidization. In males - in sharp contrast - dmrt1 was not essential for fertility and spermatogenesis in the Xenopus ancestor, but this essentiality was acquired (neofunctionalized) in the other X. laevis homeolog (dmrt1.L) after allotetraploidization. Transcriptomic analysis of the mesonephros/gonad complex during sexual differentiation identifies distinctive patterns of dysregulation in male and female knockouts of dmrt1.L and dmrt1.S relative to same-sex wildtype siblings, including possible autocatalysis of dmrt1.L and activation of the female-determining gene dm-w. Previous work demonstrates that dm-w was recently derived from partial gene duplication of dmrt1.S - a gene that our analysis demonstrates is non-essential in both sexes. Thus, in X. laevis, a developmental system was pushed past a "tipping point" to a novel state where sexual differentiation is now orchestrated by a sex-specific duplicate of a dispensable gene.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive bilateral cyst formation. Multiple cellular pathways including second messenger cAMP signaling are dysregulated in ADPKD, but mechanisms initiating cysts are unknown. ADPKD is caused by mutations in PKD1/PKD2 genes encoding for polycystins that localize to primary cilia-nonmotile, microtubule-based dynamic compartments sensing extracellular chemical/mechanical signals. The compact cylindrical structure of cilia enables tunable signaling amplification regulatable by ciliary length. Severe cystogenesis from polycystin loss is cilia dependent and ciliary elongation is common in cystic epithelia. However, uncoupling the cilium-specific signals repressed by polycystins from downstream cystogenic pathways has proven challenging. Here we aim to understand roles of compartmentalized cAMP signaling in cystogenesis and ciliary length control. We investigated ANKMY2, an Ankyrin repeat MYND domain protein involved in maturation and ciliary localization of membrane adenylyl cyclases-enzymes generating cAMP. In kidney-specific Ankmy2/Pkd1 knockout mice, loss of ANKMY2 suppressed early postnatal cystogenesis and significantly extended survival in an embryonic-onset Pkd1 deletion model. Similarly, in an adult inducible Pkd1 knockout model, ANKMY2 deficiency reduced cyst burden. Mechanistically, ANKMY2 controlled the ciliary trafficking of multiple adenylyl cyclases in mouse and human kidney epithelial cells without disrupting cilia while retaining cellular pools. Ciliary elongation began in dilatated tubules of adult onset ADPKD mice and further increased in cystic kidneys. Both initial and progressive phases of cilia lengthening were ANKMY2-dependent. Our findings indicate that ciliary adenylyl cyclase signaling likely promotes cilia-dependent cyst initiation distinct from cyst progression involving cellular cAMP. Importantly, kidneys lacking ANKMY2 did not show ciliary elongation despite elevated cAMP, suggesting that cilia lengthening during cyst progression could be contingent upon pre-cystic ciliary regulation. These results suggest a critical role for compartmentalized adenylyl cyclase signaling in ADPKD pathogenesis and a framework for identifying ciliary effectors and early subcellular events in cystogenesis.