PLoS Genetics最新文献

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.

Gene amplification is thought to be common in bacterial populations, providing a rapid and reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen Staphylococcus aureus evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via amplification of an efflux pump encoding gene sdrM. However, the pathways that control gene amplification, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like S. aureus. Here, we show that specific DNA repair and chromosomal separation proteins alter the frequency of gene amplification and selection of amplified regions in S. aureus. Through a screen of 40 mutants deficient in various DNA processes, we determined that while sdrM amplification was still the almost universal path to DLX resistance, other mutations that increased sdrM expression reduced the selection frequency of sdrM amplification, demonstrating the critical role of sdrM in DLX resistance. We found that similar to other bacteria, both sdrM amplification and loss of amplified gene copies required a functional RecA recombinase, but multiple other mutants in pathways required for amplification in other species still exhibited frequent sdrM amplification, suggesting that S. aureus may have alternate routes of gene amplification. Finally, loss of function mutants of the tyrosine recombinase XerC, that is known to play a role in chromosomal separation, were deficient for sdrM amplification, indicating that XerC is a novel modulator of gene amplification, or the maintenance or selection of amplified gene copies. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in S. aureus and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.

Protein palmitoylation in the Golgi apparatus is critical for the appropriate sorting of various proteins belonging to secretory and lysosomal systems, and defective palmitoylation can lead to the onset of severe pathologies. HIP14 and HIP14L ankyrin repeat-containing palmitoyl transferases were linked to the pathogenesis of Huntington's disease, however, how perturbation of these Golgi resident enzymes contributes to neurological disorders is yet to be understood. In this study, we investigated the function of Hip14 and Patsas - the Drosophila orthologs of HIP14 and HIP14L, respectively - to uncover their role in secretory and lysosomal membrane trafficking. Using larval salivary gland, a well-established model of the regulated secretory pathway, we found that these PAT enzymes equally contribute to the proper maturation and crinophagic degradation of glue secretory granules by mediating their fusion with the endo-lysosomal compartment. We also revealed that Patsas and Hip14 are both required for lysosomal acidification and biosynthetic transport of various lysosomal hydrolases, and we demonstrated that the rate of secretory granule-lysosome fusion and subsequent acidification positively correlates with the level of Hip14. Furthermore, Hip14 is also essential for proper lysosome morphology and neuronal function in adult brains. Finally, we found that the over-activation of lysosomal biosynthetic transport and lysosomal fusions by the expression of the constitutively active form of Rab2 could compensate for the lysosomal dysfunction caused by the loss of Patsas or Hip14 both in larval salivary glands and neurons. Therefore, we propose that ankyrin repeat palmitoyl transferases act as rate-limiting factors in lysosomal fusions and provide genetic evidence that defective protein palmitoylation and the subsequent lysosomal dysfunction can contribute to the onset of Huntington's disease-like symptoms.

Deciphering the molecular basis of complex traits requires understanding how natural genetic variation interacts with underlying biological pathways. In this study, we explored how natural genetic variation influences traits in maize affected by a semi-dominant maize dwarfing allele, Dwarf13-1 (D13-1) which encodes a defective ionotropic glutamate receptor (GLR). This allowed us to investigate natural genetic variation in the genome affecting GLR signaling in maize. We implemented an F1 association mapping (FOAM) approach, where heterozygous mutants carrying the semi-dominant D13-1 allele were crossed with a maize association panel. The resulting F1 families segregated 1:1 for mutant and wild-type phenotypes allowing comparisons between the congenic F1 hybrid siblings to identify and map natural alleles that interact with the D13-1 mutant allele. FOAM mapping detected two loci that modify the expression of the D13-1/+ mutant phenotype. The phenotypic impacts of both loci were epistatically controlled by D13-1, and only affected the phenotypes of mutant F1 hybrids. One, tropotriskaideka1 (tod1), encoded a maize homolog of the GLR-interacting cornichon gene and modified D13-1/+ mutant severity. A second, encoded by the d13 locus itself, affected the severity of the D13-1/+ phenotype via variation in the wild-type allele in the heterozygous mutants. By integrating gene expression analyses, these epistatic interactions, and SNP linkage information we identified multiple, unlinked, alleles affecting expression of the wild-type D13 transcript that modify mutant trait expression. Greater expression of the wild-type D13 allele increased plant height and suppressed D13-1/+ mutant severity, consistent with a multi-subunit complex GLR structure and complex-poisoning mode-of-action for the semi-dominant D13-1 allele. This approach identifies natural alleles affecting the GLR pathway in maize and establishes GLRs and their interactors as dose-dependent regulators of plant architecture. Our pathway-focused framework and epistasis testing of natural variants provides greater confidence in identifying genes contributing to complex traits.

Regulation of peptidoglycan hydrolases is crucial for bacterial cell integrity, growth and division. In the bacterial pathogen Staphylococcus aureus, the amidase Sle1 is a key autolysin required for septum splitting and daughter cell separation. Through genetic suppressor screening, we have identified CxaR, a previously uncharacterized protein, as a novel negative regulator of Sle1. In the absence of CxaR, cellular levels of Sle1 increase nearly ten-fold, resulting in premature splitting of the division septum and increased cell lysis during exponential growth. CxaR localizes to the division septum, late in septum synthesis, and this localization requires both the divisome protein FtsK and the ClpX component of the ClpXP proteolytic machinery. We propose that CxaR promotes ClpXP-mediated degradation of Sle1 towards the end of the cell cycle.

Prostate cancer exhibits a strong familial association, and its heritability indicates a significant contribution from germline variants. While genome-wide association studies (GWAS) have identified common germline variants associated with prostate cancer risk, translating these statistical associations into functional mechanisms has remained a long-standing challenge. Consequently, most of our understanding of the genetic basis of prostate cancer stems from extensive studies of somatic mutations, leaving the germline genetic architecture largely unresolved. Because most germline variants lie in the noncoding genome and complex human diseases are predominantly driven by regulatory mutations, we herein asked which prostate cell types mediate the functional effects of germline variants, and thus represent the most genetically vulnerable populations. We generated paired epigenomic and transcriptomic profiles from reference human prostate tissues. Integrating these single-cell data with large-scale GWAS data identified a terminally differentiated luminal epithelial subtype that mediates the strongest germline risk in prostate cancer. We subsequently developed a deep learning model to score ~17 million GWAS variants based on their predicted impact on altering local chromatin accessibility in this vulnerable luminal epithelial subtype, and identified high-confidence candidate loci where high-risk germline variants likely alter promoter accessibility in prostate cancer. The implicated genes were involved in several pathways in tumorigenesis, displayed strong dosage sensitivity, and converged on the androgen receptor (AR)-mediated regulon, a mechanism also observed for somatic mutations. Overall, by unveiling cell types and candidate loci that mediate germline risk, our study defines the cell-type-specific germline architecture in prostate cancer and provides a comprehensive framework for understanding cancer heritability.

Mendelian Randomisation Egger regression (MR-Egger) is a popular method for causal inference using single-nucleotide polymorphisms (SNPs) as instrumental variables. It allows all SNPs to have direct pleiotropic effects on the outcome, provided that those effects are independent of the effects on the exposure, known as the InSIDE assumption. However, the results of MR-Egger, and the InSIDE assumption itself, are sensitive to which allele is coded as the effect allele for each SNP. A pragmatic convention is to code the alleles with positive effects on the exposure, which has some advantages in interpretation but some statistical limitations. Here we show that if the InSIDE assumption holds under all-positive coding of the exposure effects, it cannot hold under all-positive coding of the pleiotropic effects, and argue that this undermines the soundness of MR-Egger. We propose a modification that has the Genotype Recoding Invariance Property (GRIP), achieving the main aim of MR-Egger without the difficulties of allele coding. Our approach, MR-GRIP, is valid under a "Variance Independent of Covariance Explained" assumption (VICE), which amounts to an inverse relationship between exposure effects and pleiotropic effects. Examples and simulations suggest that MR-GRIP can reconcile differences between MR-Egger and alternative methods.

Streptococcus pneumoniae is a pathogenic bacterium capable of entering a cellular differentiation state, called competence, which enables it to acquire new genetic functions by natural transformation, as well as physiological functions such as tolerance to a number of antibiotics. The transition to this state is regulated by various environmental or intracellular signals that converge on the comCDE operon, which groups together the competence initiation genes. A fraction of activated cells is sufficient to propagate competence to the whole population via the product of the comC gene, the competence stimulating peptide (CSP). Remarkably, depletion of the essential ClpX/ ClpP AAA+ protease has been shown to induce the comCDE operon. Here we demonstrate that the ClpX-dependent induction of competence relies on the Spr1630 toxin (RipA), part of a Rosmer toxin-antitoxin system. We show that this toxin generates replicative stress by acting on the sliding clamp of replication, inducing transcription of the comCDE operon. Bacteria that produce RipA appear to lose their viability but remain metabolically active and able to produce CSP, thereby transferring competence to viable neighbouring cells.

The distinct textural properties of fruits in varying stages of ripening present unique ecological opportunities for several species of fruit flies, resulting, over evolutionary times, in specialized egg-laying behaviors. In this study we identified a TrpA channel-dependent mechanosensory pathway in the legs, through the gene painless, that modulates the discernment of softer patches for oviposition in gravid D. melanogaster females. We report that the stiffness-sensing role of tarsi is mediated through external sensory organs, namely ventral mechanosensory bristles and subsets of campaniform sensilla present primarily at the joints between tarsomeres. Our findings provide new evidence that campaniform sensilla function as indirect stiffness sensors of oviposition substrates, owing to their placement at joints that experience maximal cuticular distortion. We show that Painless is expressed in mechanosensory neurons innervating peripheral organs and is necessary for their functions in mediating oviposition substrate selection in gravid females. Furthermore, we observed that overexpression of painless in both campaniform sensilla and mechanosensory bristles partially rescues preference for the softer substrates in painless mutants, indicating that painless activity in these organs is necessary to mediate the preference. We propose that different interactions with a soft vs. a hard substrate (compression of the cuticle, distribution of contacts) results in differential mechanotransduction in painless-expressing neurons, determining oviposition preferences.

A key pathological feature of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) is the loss of nuclear localization and accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43). TDP-43 is a nucleic acid-binding protein involved in transcriptional repression, mRNA splicing, and the regulation of retrotransposable elements (RTEs) and endogenous retroviruses (ERVs). RTEs/ERVs are mobile virus-like genetic elements that constitute about 45% of our genome and encode the capacity to replicate through an RNA intermediate and insert cDNA copies at de novo chromosomal locations. A causal role of RTEs/ERVs has been demonstrated in Drosophila in mediating both intracellular toxicity of TDP-43 and the intercellular spread of toxicity from glia to neurons. RTEs/ERVs are inappropriately expressed in postmortem tissues from ALS, FTD, and Alzheimer's Disease (AD) patients, but the role of RTEs/ERVs has not yet been examined in a vertebrate model of TDP-43 pathology. We utilized established transgenic mouse models that overexpress moderate levels of human wild-type TDP-43 or a mutant version with a specific ALS-causal Q331K amino acid substitution, together with a LINE-1-EGFP retrotransposon indicator line. We found that TDP-43 animals exhibit broad expression of RTEs/ERVs with LINE-1 retrotransposition in glia and neurons in the motor cortex. Expression begins with onset of neurological phenotypes, earlier in hTDP-43-Q331K animals and later in hTDP-43-WT. The LINE-1-EGFP retrotransposition reporter transiently labels spatially clustered groups of neurons and glia at the time of onset of motor symptoms, while EGFP-labeled neurons undergo cell death and are therefore lost over time. Unlabeled cells also die as a function of distance from the clusters of LINE-1-EGFP labeled neurons and glial cells. Together, these findings support the hypothesis that TDP-43 pathology triggers RTE/ERV expression in the motor cortex, that such expression marks cells for programmed cell death, with cell non-autonomous effects on nearby neurons and glial cells.