PLoS Genetics最新文献

Mutations in mitochondrial DNA (mtDNA) can lead to mitochondrial and cellular dysfunction. However, recent studies suggest that purifying selection acts against mutant mtDNAs during transgenerational transmission. We investigated the mtDNA dynamics during ovarian follicle development. Using base-editing, we generated mice harboring a 3177 G > A mutation corresponding to the human Leber hereditary optic neuropathy (LHON)-related mtDNA mutation and confirmed a transgenerational reduction of the mutant mtDNA. Utilizing a mouse follicle culture system in which pathogenic mtDNA mutations were introduced in vitro, followed by mtDNA sequencing and digital PCR, we found that the germline heteroplasmy shift during early folliculogenesis was driven by a decrease in mutant mtDNA along with compensatory replication of wild-type mtDNA. In contrast, synonymous mtDNA mutations did not affect mtDNA dynamics. These findings demonstrate that mice can eliminate certain pathogenic mtDNA mutations in the germline during early folliculogenesis, thus advancing our understanding of mtDNA purifying selection during oogenesis. Furthermore, our use of mtDNA editing in in vitro-cultured follicles provides a novel approach to create and monitor mitochondrial DNA mutations.

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression. Some are essential for organismal development and physiology, and they can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma that have positionally equivalent transcripts in zebrafish. We prioritized the cancer-associated lncRNA Differentiation Antagonizing Non-Protein Coding RNA (DANCR) as an exemplar for functional investigation. DANCR is a multi-exonic, cytoplasmically-enriched lncRNA and small RNA host gene transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks to promote human melanoma cell proliferation and migration. Zebrafish dancr is essential for embryonic development. It is dynamically expressed across multiple different cell types in the developing embryo, transcriptionally activated by mitfa during early zebrafish development and it regulates genes involved in cell death. Our work suggests that cancer-critical lncRNAs such as DANCR, expressed from similar regions in vertebrate genomes, may control related genes and processes involved in both embryonic development and tumorigenesis across species.

Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded (GGCCTG)n hexanucleotide repeat sequence in the NOP56 gene. While the expanded repeats could transcribe and form toxic RNA foci within neurons, recent evidence indicates that translation of these repeats produces dipeptide repeats (DPR) that contribute to neurotoxicity. The relative impact of hexanucleotide RNA repeats (HRR) and DPR on the neurodegeneration of SCA36 remains unclear. Here, we established a Drosophila SCA36 model to dissect the neurotoxic effects of HRR and DPR. The fly model recapitulates the cellular defects observed in SCA36 patient fibroblasts, validating its relevance for mechanistic study of SCA36. Further engineering the transgenes to express individual DPRs reveal Proline-Glycine-DPR (PG-DPR) as the most potent neurotoxin causing progressive motor and sensory dysfunction. Expressing a series of the SCA36 transgenes with varying HRR lengths demonstrates an age- and length-dependent adult-onset neurodegeneration. Interestingly, sequence modification of the transgenes to exclusively express HRR or DPR alone causes a milder phenotype, indicating both HRR and DPR contribute partially to the pathogenicity of SCA36. Therefore, this model provides a valuable platform for screening drug targeting either HRR- or DPR-mediated toxicity of SCA36. Suppression of the RNA elongation factor SUPT4H1 ortholog reduces RNA foci in cell culture. However, expression level of SUPT4H1 was not changed in SCA36 patient cells. Interestingly, knockdown of the Drosophila SUPT4H1 ortholog or 6-azauridine treatment to suppress RNA transcription aggravates the neurodegenerative phenotypes in both the fly models and patient-derived fibroblasts, highlighting the complex interplay of pathomechanisms in SCA36. These results underscore the need for carefully evaluating the potential side effects when designing therapeutic interventions for SCA36.

Early life experiences such as malnutrition can affect development and adult disease risk, but the molecular basis of such protracted effects is poorly understood. In the nematode C. elegans, extended starvation during the first larval stage causes the development of germline tumors and other abnormalities in the adult gonad, limiting reproductive success. Insulin/IGF signaling (IIS) acts through WNT signaling and lipid metabolism to promote starvation-induced gonad abnormalities, but IIS-independent modifiers have not been identified. The tumor suppressor daf-18/PTEN inhibits IIS to suppress starvation-induced abnormalities, but we show that it also acts independently of IIS via lin-35/Rb, another tumor suppressor, to suppress such abnormalities. We found that lin-35/Rb and the rest of the DREAM complex repress transcription of the Hedgehog (Hh) signaling homologs ptr-23/PTCH-related, wrt-1/Hh-like, and wrt-10/Hh-like, which promote starvation-induced abnormalities. These Hh-related genes transcriptionally activate several genes associated with innate immunity in adults, which also promote starvation-induced gonad abnormalities. Surprisingly, we found that in addition to causing developmental abnormalities, early-life starvation induces an innate immune response later in life, leading to increased resistance to bacterial and intracellular pathogens. This work identifies a critical tumor-suppressor function of daf-18/PTEN independent of IIS, and it defines a regulatory network, including lin-35/Rb and DREAM, Hh-related signaling, and innate immunity pathways, that affects development of tumors and other developmental abnormalities resulting from early life starvation. By revealing that early-life starvation increases immunity later in life, this work suggests a fitness tradeoff between pathogen resistance and developmental robustness.

Inherited retinal diseases (IRDs) are a diverse group of disorders that share common vision deficits ranging from early onset blindness to severe and progressive later-onset disease. We report a form of early-onset day-vision loss, cone-rod dystrophy, in the Standard poodle. Through GWAS and homozygosity mapping, a large deletion on CFA8:NC_049229.1:g.60,022,583_60,040,453del was found which removes 3' portions of two different genes, PTPN21 and SPATA7, presenting a challenge for assessing the actual causative gene in a multi-gene large deletion. All affected dogs were homozygous for the mutant allele, which segregated perfectly with the phenotype within the breed. The variant was absent in 1879 dogs from the Dog10K database. While the role of SPATA7 for retinal disease has been established in human patients and genetically engineered mice, the role of PTPN21 in the retina is unclear even though it is expressed in rod and cone photoreceptors. Expression of whole and truncated transcripts for both genes was detected in skin fibroblasts from controls and cases. Retinal RNA analysis of PTPN21 splicing suggests that at least one unmodified transcript is still present in mutants. Ptpn21-/- knockout mice did not have an ocular phenotype, and IHC for rod- and cone-specific opsins detected no cone or rod abnormalities suggesting that PTPN21 loss has minimal to no contributory role towards the retinal phenotype in mutants. The variant leads to a deletion of the 3'-end of the SPATA7 transcript: XM_038545497.1:r.1,314_1,629delins[g.60,018,954-60,018,990], p.(XP_038401425.1: Asp361GlufsTer2), reducing the predicted protein from 595 to 361 AA. Ultrastructure expansion microscopy (U-ExM) enabled the detection of a distinct SPATA7 signal around the transition zone of the primary cilium in photoreceptors and fibroblasts of WT dogs, which was absent in affected dog. We posit that SPATA7 deficiency is the main cause of the condition, and propose this disease as a model for the SPATA7-related form of cone-rod dystrophy in humans. Our work shows an example of functional refinement of a multi-gene deletion variant using a multi-technique approach.

Axon injury initiates transcriptional reprogramming that in competent cells leads to regeneration. In vertebrate neurons, DLK acts upstream of Jun, STAT and Atf3, core transcription factors that mediate regeneration. It is unclear whether these three proteins are activated independently, or whether they function in a linear cascade. To investigate relationships between these transcription factors we wished to use Drosophila as a model system as it has one ortholog of each. However, the only transcription factor linked to DLK-mediated axon regeneration (AR) in flies was Fos. Using loss of function approaches we demonstrate that Jun, STAT and Atf3 are required for Drosophila sensory axon regeneration, indicating transcriptional control of axon regeneration is broadly conserved. We next investigated temporal roles for Fos, Jun, STAT and Atf3. Only Fos is required for the early transcriptional response, which coincides with neuroprotection, and its nuclear entry and homodimerization coincide with this phase. Reduction of Jun homodimerization occurs after axon injury downstream of DLK/JNK, but independently from Fos, at a later stage associated with axon regrowth. STAT nuclear entry occurs downstream of Jun as part of this stage, is inhibited by Fos, and does not require JAK, which is dispensable for axon regeneration. Atf3 nuclear exit is in turn downstream of Fos, Jun, and STAT. Our results suggest that DLK/JNK separately activates Fos and Jun, and that Jun initiates a transcriptional cascade that includes STAT and Atf3. These two transcriptional modules control separate steps of the injury response that culminates in axon regeneration.

The self-renewal and differentiation of germline stem cells (GSCs) are tightly regulated during oogenesis. The Drosophila female germline provides a powerful model to study these regulatory mechanisms. We previously identified Sakura (also known as Bourbon/CG14545) as a crucial factor for maintenance and differentiation of GSCs and oogenesis, and demonstrated that Sakura binds to Ovarian Tumor (Otu), another essential regulator of these processes. Here, we identify MYCBP (c-Myc binding protein) as an additional essential component of this regulatory network. We show that MYCBP physically associates with itself, Sakura, and Otu, forming binary and ternary complexes including a MYCBP•Sakura•Otu complex. MYCBP is highly expressed in the ovary, and mycbp null mutant females exhibit rudimentary ovaries with germline-less and tumorous ovarioles, fail to produce eggs, and are completely sterile. Germline-specific depletion of mycbp disrupts Dpp/BMP signaling, causing aberrant expression of bag-of-marbles (bam) and leading to defective differentiation and GSC loss. In addition, mycbp is required for female-specific splicing of sex-lethal (sxl), a master regulator of sex identity determination. These phenotypes closely resemble those observed in sakura and otu mutants. Together, our findings reveal that MYCBP functions in concert with Sakura and Otu to coordinate self-renewal and differentiation of GSCs and oogenesis in Drosophila.

Genetic selection occurs at multiple stages before and during pregnancy. While parental genomes influence the probability of fertilization, the fetal genome, once established, plays a critical role in early fetal survival. However, when estimated separately, parental and fetal genetic effects may confound each other. To address this, we developed an extension of the case-parent triad design to jointly estimate the genetic contributions of the parents and the fetus. Our approach considers all offspring as carriers of the trait "fetal survival". As use of assisted reproductive technology (ART) usually reflects fertility issues, we performed separate analyses on non-ART and ART family units, hypothesizing that parental and fetal effects differ between these groups. In the Norwegian Mother, Father, and Child Cohort Study, we had access to genotypes for approximately 43,000 family triads and dyads, including 1,336 offspring conceived through ART. In the non-ART sample, we identified genome-wide significant fetal effects on fetal survival for SNPs within regions harboring genes relevant to infertility and fetal development, such as MDC1, MICB, HCP5, and NOTCH4. These effects remained significant after adjusting for parental interaction effects, confirming their origin as fetal effects. When we replicated the analysis in the ART sample, we observed partial overlap in fetal effects with those identified in the non-ART sample. Parental interaction effects were observed in both the non-ART and ART samples, but the specific genetic associations differed between the groups. Notably, several SNPs associated with parental interaction effects in the ART sample mapped to genes previously implicated in male infertility, including ACTB, FSCN1, and RNF216. Our findings have broad implications for understanding the genetic architecture of infertility and fetal development. To support the interpretation of our results, we provide detailed descriptions of the models, highlighting their strengths and limitations.

In plants, RNA-directed DNA methylation (RdDM) sequence-specifically targets transposable elements (TEs) and repeats, often in a tissue-specific manner. In triploid endosperm tissue, RdDM also acts as a parental dosage regulator, mediating spatio-temporal expression of genes required for its development. It is unclear how RdDM is initiated and established in endosperm. Rice endosperm-specific imprinted chromatin remodeler OsCLSY3 recruits RNA polymerase IV to specific genomic sites for silencing and optimal gene expression. Here we show that, in addition to OsCLSY3, ubiquitously expressed OsCLSY4 is also crucial for proper reproductive growth and endosperm development. Loss of function of OsCLSY4 led to reproductive and nutrient-filling defects in endosperm. Using genetic and molecular analysis, we show that both OsCLSY3 and OsCLSY4 play overlapping and unique silencing roles in rice endosperm, by targeting specific and shared genomic regions such as TEs, repeats and genic regions. These results indicate the importance of optimal expression of two OsCLSYs in regulating endosperm-specific gene expression, genomic imprinting and suppression of specific TEs. Results presented here provide new insights into the functions of rice CLSYs as upstream RdDM regulators in rice endosperm development, and we propose that functions of their homologs might be conserved across monocots.

Zinc is essential for many physiological processes and its deficiency is highly prevalent worldwide. Its complex homeostasis involves membrane transporters from the SLC39/ZIP and SLC30/ZnT protein families. We conducted a genome-wide association study (GWAS) meta-analysis of urinary zinc levels in three European-ancestry cohorts (N = 10,113), followed by in silico and in vivo studies to elucidate their underlying public health and physiological relevance. We identified eleven genome-wide significant signals with six mapping to SLC39/ZIP and SLC30/ZnT gene regions. The lead signal (rs3008217C>G, p = 2.42E-110) in the SLC30A2 gene region which explained 6.1% of urinary zinc variation strongly colocalized with its expression in kidney tubules. Low phenotypic and genetic correlations between plasma and urinary zinc levels indicated distinct genetic regulation. High urinary zinc correlated with an unfavorable cardiometabolic profile, and Mendelian randomization analyses suggested causal roles for diabetes increasing urinary zinc levels, and elevated urinary zinc increasing stroke risk. Analyzing country-level allele frequencies and zinc deficiency prevalences revealed a 3-fold higher genetic zinc excretion risk in sub-Saharan Africa compared to Europe, significantly correlating with nutritional zinc deficiency prevalence. Although mutations in SLC30A2 are linked to insufficient zinc in human milk, we found no association with common variants using data generated from 387 mothers. Mice experiments showed that dietary zinc deficiency decreased urinary but not plasma zinc levels, and upregulated kidney Slc30a2 expression. This first GWAS on urinary zinc highlights the involvement of zinc transporters in its genetic regulation, as well as its role as a non-invasive biomarker for cardiometabolic diseases.