ABSTRACT
BACKGROUND: The remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes. RESULTS: Here, we ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions. CONCLUSIONS: These findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.
Subject(s)
Regeneration , Animals , Regeneration/genetics , Regeneration/physiology , Aging/genetics , Aging/physiology , Species Specificity , Drosophila/genetics , Drosophila/physiology , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Drosophila melanogaster/growth & development , Stem Cells/metabolism , Intestines/physiology , Cell Differentiation/genetics , Cell ProliferationABSTRACT
The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: in MicroC experiments, the eve TAD is a 'volcano' triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the 'new' eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is converted to a pair of 'clouds' of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.
Subject(s)
Drosophila Proteins , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Drosophila melanogaster/genetics , Drosophila/geneticsABSTRACT
Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.
Subject(s)
Drosophila , Animals , Drosophila/genetics , Drosophila melanogaster/genetics , Chromatin/chemistry , Chromatin/metabolism , Cohesins , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosome Structures , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/chemistryABSTRACT
Similar to the physiological importance of gut microbiomes, recent works have shown that insect ectomicrobiotas can mediate defensive colonization resistance against fungal parasites that infect via cuticle penetration. Here we show that engineering the entomopathogenic fungus Metarhizium robertsii with a potent antibacterial moricin gene from silkworms substantially enhances the ability of the fungus to kill mosquitos, locusts, and two Drosophila species. Further use of Drosophila melanogaster as an infection model, quantitative microbiome analysis reveals that engineered strains designed to suppress insect cuticular bacteria additionally disrupt gut microbiomes. An overgrowth of harmful bacteria such as the opportunistic pathogens of Providencia species is detected that can accelerate insect death. In support, quantitative analysis of antimicrobial genes in fly fat bodies and guts indicates that topical fungal infections result in the compromise of intestinal immune responses. In addition to providing an innovative strategy for improving the potency of mycoinsecticides, our data solidify the importance of both the ecto- and endo-microbiomes in maintaining insect wellbeing.
Subject(s)
Metarhizium , Animals , Metarhizium/genetics , Drosophila melanogaster/microbiology , Drosophila melanogaster/genetics , Bombyx/microbiology , Bombyx/genetics , Pest Control, Biological/methods , Gastrointestinal MicrobiomeABSTRACT
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , RNA Polymerase II , Transcription, Genetic , Transcriptional Elongation Factors , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Positive Transcriptional Elongation Factor B/metabolism , Positive Transcriptional Elongation Factor B/genetics , Promoter Regions, Genetic , CRISPR-Cas Systems , Transcription Factors/metabolism , Transcription Factors/genetics , Polytene Chromosomes/genetics , Polytene Chromosomes/metabolism , Gene Expression Regulation , Phosphorylation , Protein Binding , Heat-Shock Response/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Nucleosomes/metabolism , Nucleosomes/geneticsABSTRACT
The transmembrane protein ß-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The ß-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic ß-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.
Subject(s)
Amyloid beta-Protein Precursor , Drosophila Proteins , Neuromuscular Junction , rab GTP-Binding Proteins , Animals , Neuromuscular Junction/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Protein Precursor/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Synaptic Transmission , Synapses/metabolism , Receptors, Glutamate/metabolism , Receptors, Glutamate/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Humans , DNA Helicases/metabolism , DNA Helicases/genetics , Membrane Proteins , Nerve Tissue Proteins , Homeodomain Proteins , Receptors, Ionotropic GlutamateABSTRACT
RHOBTB2 was first described as epileptogenic when it presents a missense variant in 2016 and studied more specifically in 2018. It is a gene that causes rare, but potentially severe childhood epileptic encephalopathy. In 2021, research confirmed that heterozygous mutations of RHOBTB2 included other clinical signs besides these encephalopathies. Thus, these infantile epilepsies are mainly associated with highly variable phenotypes, with developmental delay, post-traumatic encephalitis, paroxysmal movement disorders and iconographic brain damage. In this work, after presenting a clinical case, we will recall the role of RhoGTPases on neuronal development. We will then discuss a study which highlighted the neurodevelopmental impact of mutations on the RHOBTB2 gene by carrying out work on Drosophila melanogaster flies. Finally, we will compare the presented clinical case with a literature review.
Le gène RHOBTB2 est décrit pour la première fois comme épileptogène alors qu'il présente un variant faux-sens en 2016, puis est étudié plus précisément en 2018. Il s'agit d'un gène qui est à l'origine d'encéphalopathies épileptiques infantiles rares, mais pouvant être sévères. En 2021, des recherches ont confirmé que les mutations hétérozygotes de RHOBTB2 englobaient d'autres signes cliniques que ces encéphalopathies. Ainsi, ces épilepsies infantiles sont associées, principalement, avec des phénotypes fortement variables, à un retard développemental, à des encéphalites post-traumatiques, à des troubles paroxystiques des mouvements et à des atteintes iconographiques de l'encéphale. Dans ce travail, après avoir présenté un cas clinique, nous rappellerons le rôle des RhoGTPases sur le développement neuronal. Nous discuterons ensuite d'une étude qui a mis en évidence l'impact neurodéveloppemental de mutations sur le gène RHOBTB2 en réalisant des travaux sur des mouches Drosophila melanogaster. Pour terminer, nous mettrons le cas clinique présenté en parallèle avec une revue de la littérature réalisée par rapport à ce gène.
Subject(s)
Mutation , Humans , Animals , Tumor Suppressor Proteins/genetics , GTP-Binding Proteins/genetics , Male , Drosophila melanogaster/genetics , Female , InfantABSTRACT
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
Subject(s)
Carcinogenesis , Drosophila Proteins , Proteolysis , Ubiquitin-Protein Ligases , Animals , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Carcinogenesis/genetics , Humans , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/embryology , Genes, Tumor Suppressor , Ubiquitination , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 1/geneticsABSTRACT
The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.
Subject(s)
DNA-Binding Proteins , Drosophila Proteins , Drosophila melanogaster , ErbB Receptors , Gene Expression Regulation, Developmental , Neurons , Signal Transduction , Transcription Factors , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , ErbB Receptors/metabolism , ErbB Receptors/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Neurons/metabolism , Neurons/cytology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Differentiation , Photoreceptor Cells, Invertebrate/metabolism , Photoreceptor Cells, Invertebrate/cytology , Eye Proteins/metabolism , Eye Proteins/genetics , Imaginal Discs/metabolism , Imaginal Discs/cytology , Nerve Tissue Proteins , Proto-Oncogene Proteins , Receptors, Invertebrate PeptideABSTRACT
Sex chromosomes underlie the development of male or female sex organs across species. While systemic signals derived from sex organs prominently contribute to sex-linked differences, it is unclear whether the intrinsic presence of sex chromosomes in somatic tissues has a specific function. Here, we use genetic tools to show that cellular sex is crucial for sexual differentiation throughout the body in Drosophila melanogaster. We reveal that every somatic cell converts the intrinsic presence of sex chromosomes into the active production of a sex determinant, a female specific serine- and arginine-rich (SR) splicing factor. This discovery dismisses the mosaic model which posits that only a subset of cells has the potential to sexually differentiate. Using cell-specific sex reversals, we show that this prevalence of cellular sex drives sex differences in organ size and body weight and is essential for fecundity. These findings demonstrate that cellular sex drives differentiation programs at an organismal scale and highlight the importance of cellular sex pathways in sex trait evolution.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Sex Chromosomes , Sex Differentiation , Animals , Male , Female , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Sex Differentiation/genetics , Sex Differentiation/physiology , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Sex Chromosomes/genetics , Fertility/genetics , Sex Characteristics , Organ Size , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , Body Weight , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.
Subject(s)
CRISPR-Cas Systems , Drosophila Proteins , Eye Color , Gene Editing , Animals , Gene Editing/methods , Drosophila Proteins/genetics , Eye Color/genetics , Animals, Genetically Modified , Transcription Factors/genetics , Drosophila/genetics , Students , Drosophila melanogaster/genetics , RNA, Guide, CRISPR-Cas Systems/genetics , Eye Proteins , ATP-Binding Cassette TransportersABSTRACT
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
Subject(s)
Cytosol , Drosophila Proteins , Endocytosis , Lysosomes , RNA Interference , RNA, Double-Stranded , Animals , RNA, Double-Stranded/metabolism , Lysosomes/metabolism , Cytosol/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Chloride Channels/metabolism , Chloride Channels/genetics , Cell Line , Intracellular Membranes/metabolism , Permeability , Drosophila/metabolism , Drosophila/geneticsABSTRACT
Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.
Subject(s)
Biomolecular Condensates , Drosophila Proteins , Drosophila melanogaster , Hippo Signaling Pathway , Neurofibromin 2 , Animals , Cell Membrane/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Intercellular Junctions/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Neurofibromin 2/metabolism , Neurofibromin 2/genetics , Phosphatidylinositol Phosphates/metabolism , Protein Serine-Threonine Kinases/metabolism , Biomolecular Condensates/metabolismABSTRACT
Ribosomes are regulated by evolutionarily conserved ubiquitination/deubiquitination events. We uncover the role of the deubiquitinase OTUD6 in regulating global protein translation through deubiquitination of the RPS7/eS7 subunit on the free 40 S ribosome in vivo in Drosophila. Coimmunoprecipitation and enrichment of monoubiquitinated proteins from catalytically inactive OTUD6 flies reveal RPS7 as the ribosomal substrate. The 40 S protein RACK1 and E3 ligases CNOT4 and RNF10 function upstream of OTUD6 to regulate alkylation stress. OTUD6 interacts with RPS7 specifically on the free 40 S, and not on 43 S/48 S initiation complexes or the translating ribosome. Global protein translation levels are bidirectionally regulated by OTUD6 protein abundance. OTUD6 protein abundance is physiologically regulated in aging and in response to translational and alkylation stress. Thus, OTUD6 may promote translation initiation, the rate limiting step in protein translation, by titering the amount of 40 S ribosome that recycles.
Subject(s)
Drosophila Proteins , Protein Biosynthesis , Ribosomal Proteins , Ubiquitination , Animals , Ribosomal Proteins/metabolism , Ribosomal Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Ribosomes/metabolism , Stress, Physiological , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.
Subject(s)
Disease Models, Animal , Drosophila Proteins , GTP Phosphohydrolases , Optic Atrophy, Autosomal Dominant , Animals , Optic Atrophy, Autosomal Dominant/genetics , Optic Atrophy, Autosomal Dominant/metabolism , Optic Atrophy, Autosomal Dominant/pathology , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Humans , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Mutation , Drosophila/genetics , Membrane ProteinsABSTRACT
Understanding the evolutionary potential of mutations in gene regulatory networks is essential to furthering the study of evolution and development. However, in multicellular systems, genetic manipulation of regulatory networks in a targeted and high-throughput way remains challenging. In this study, we designed TF-High-Evolutionary (HighEvo), a transcription factor (TF) fused with a base editor (activation-induced deaminase), to continuously induce germline mutations at TF-binding sites across regulatory networks in Drosophila. Populations of flies expressing TF-HighEvo in their germlines accumulated mutations at rates an order of magnitude higher than natural populations. Importantly, these mutations accumulated around the targeted TF-binding sites across the genome, leading to distinct morphological phenotypes consistent with the developmental roles of the tagged TFs. As such, this TF-HighEvo method allows the interrogation of the mutational space of gene regulatory networks at scale and can serve as a powerful reagent for experimental evolution and genetic screens focused on the regulatory genome.
Subject(s)
Gene Regulatory Networks , Transcription Factors , Animals , Transcription Factors/genetics , Transcription Factors/metabolism , Genome, Insect , Mutagenesis , Drosophila/genetics , Evolution, Molecular , Drosophila melanogaster/geneticsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Kinesins , Motor Neurons , Animals , Kinesins/genetics , Kinesins/metabolism , Motor Neurons/metabolism , Motor Neurons/pathology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/metabolism , Mutation , Humans , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila , Neuromuscular Junction/metabolism , Neuromuscular Junction/pathology , Drosophila melanogaster/genetics , Synaptic Transmission/genetics , Disease Models, Animal , Axons/metabolism , Axons/pathology , Larva/genetics , Larva/metabolismABSTRACT
SLC30A9 (ZnT9) is a mitochondria-resident zinc transporter. Mutations in SLC30A9 have been reported in human patients with a novel cerebro-renal syndrome. Here, we show that ZnT9 is an evolutionarily highly conserved protein, with many regions extremely preserved among evolutionarily distant organisms. In Drosophila melanogaster (the fly), ZnT9 (ZnT49B) knockdown results in acutely impaired movement and drastic mitochondrial deformation. Severe Drosophila ZnT9 (dZnT9) reduction and ZnT9-null mutant flies are pupal lethal. The phenotype of dZnT9 knockdown can be partially rescued by mouse ZnT9 expression or zinc chelator TPEN, indicating the defect of dZnT9 loss is indeed a result of zinc dyshomeostasis. Interestingly, in the mouse, germline loss of Znt9 produces even more extreme phenotypes: the mutant embryos exhibit midgestational lethality with severe development abnormalities. Targeted mutagenesis of Znt9 in the mouse brain leads to serious dwarfism and physical incapacitation, followed by death shortly. Strikingly, the GH/IGF-1 signals are almost non-existent in these tissue-specific knockout mice, consistent with the medical finding in some human patients with severe mitochondrial deficiecny. ZnT9 mutations cause mitochondrial zinc dyshomeostasis, and we demonstrate mechanistically that mitochondrial zinc elevation quickly and potently inhibits the activities of respiration complexes. These results reveal the critical role of ZnT9 and mitochondrial zinc homeostasis in mammalian development. Based on our functional analyses, we finally discussed the possible nature of the so far identified human SLC30A9 mutations.
Subject(s)
Cation Transport Proteins , Embryonic Development , Mitochondria , Zinc , Animals , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Humans , Zinc/metabolism , Mice , Mitochondria/metabolism , Embryonic Development/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Evolution, Molecular , Mice, Knockout , Amino Acid Sequence , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Transcription Factors , Cell Cycle ProteinsABSTRACT
Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.
Subject(s)
Drosophila Proteins , Ecdysone , Gene Expression Regulation, Developmental , Histones , Larva , Animals , Histones/metabolism , Acetylation , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Ecdysone/metabolism , Larva/metabolism , Larva/growth & development , Larva/genetics , Bombyx/metabolism , Bombyx/genetics , Bombyx/growth & development , Juvenile Hormones/metabolism , Methylation , Drosophila melanogaster/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/genetics , Signal Transduction , Pupa/metabolism , Pupa/growth & development , Pupa/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Histone Deacetylases/metabolism , Histone Deacetylases/genetics , Insect Proteins/metabolism , Insect Proteins/genetics , Repressor Proteins , Basic Helix-Loop-Helix Transcription FactorsABSTRACT
FOXO transcription factors modulate aging-related pathways and influence longevity in multiple species, but the transcriptional targets that mediate these effects remain largely unknown. Here, we identify an evolutionarily conserved FOXO target gene, Oxidative stress-responsive serine-rich protein 1 (OSER1), whose overexpression extends lifespan in silkworms, nematodes, and flies, while its depletion correspondingly shortens lifespan. In flies, overexpression of OSER1 increases resistance to oxidative stress, starvation, and heat shock, while OSER1-depleted flies are more vulnerable to these stressors. In silkworms, hydrogen peroxide both induces and is scavenged by OSER1 in vitro and in vivo. Knockdown of OSER1 in Caenorhabditis elegans leads to increased ROS production and shorter lifespan, mitochondrial fragmentation, decreased ATP production, and altered transcription of mitochondrial genes. Human proteomic analysis suggests that OSER1 plays roles in oxidative stress response, cellular senescence, and reproduction, which is consistent with the data and suggests that OSER1 could play a role in fertility in silkworms and nematodes. Human studies demonstrate that polymorphic variants in OSER1 are associated with human longevity. In summary, OSER1 is an evolutionarily conserved FOXO-regulated protein that improves resistance to oxidative stress, maintains mitochondrial functional integrity, and increases lifespan in multiple species. Additional studies will clarify the role of OSER1 as a critical effector of healthy aging.