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In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.
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Citoesqueleto de Actina , Proteínas de Drosophila , Integrinas , Podócitos , Animais , Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Integrinas/metabolismo , Podócitos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila/metabolismo , Actinas/metabolismo , ImunoglobulinasRESUMO
Strain differences have been reported for motor behaviors, and only a subset of spinal cord injury (SCI) patients develop neuropathic pain, implicating genetic or genomic contribution to this condition. Here, we evaluated neuropsychiatric behaviors in A/J, BALB/c, and C57BL/6 male mice and tested genetic or genomic alterations following SCI. A/J and BALB/c naive mice showed significantly less locomotor activity and greater anxiety-like behavior than C57BL/6 mice. Although SCI elicited locomotor dysfunction, C57BL/6 and A/J mice showed the best and the worst post-traumatic recovery, respectively. Mild (m)-SCI mice showed deficits in gait dynamics. All moderate/severe SCI mice exhibited similar degrees of anxiety/depression. mSCI in BALB/c and A/J mice resulted in depression, whereas C57BL/6 mice did not exhibit depression. mSCI mice had significantly lower mechanical thresholds than their controls, indicating high cutaneous hypersensitivity. C57BL/6, but not A/J and BLAB/c mice, showed significantly lower heat thresholds than their controls. C57BL/6 mice exhibited spontaneous pain. RNAseq showed that genes in immune responses and wound healing were upregulated, although A/J mice showed the largest increase. The cell cycle and the truncated isoform of trkB genes were robustly elevated in SCI mice. Thus, different genomics are associated with post-traumatic recovery, underscoring the likely importance of genetic factors in SCI.
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Depressão , Hiperalgesia , Locomoção , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/fisiopatologia , Hiperalgesia/genética , Locomoção/genética , Camundongos , Depressão/genética , Depressão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Especificidade da EspécieRESUMO
Highly evolutionarily conserved multiprotein complexes termed Complex of Proteins Associated with Set1 (COMPASS) are required for histone 3 lysine 4 (H3K4) methylation. Drosophila Set1, Trx, and Trr form the core subunits of these complexes. We show that flies deficient in any of these three subunits demonstrated high lethality at eclosion (emergence of adult flies from their pupal cases) and significantly shortened lifespans for the adults that did emerge. Silencing Set1, trx, or trr in the heart led to a reduction in H3K4 monomethylation (H3K4me1) and dimethylation (H3K4me2), reflecting their distinct roles in H3K4 methylation. Furthermore, we studied the gene expression patterns regulated by Set1, Trx, and Trr. Each of the COMPASS core subunits controls the methylation of different sets of genes, with many metabolic pathways active early in development and throughout, while muscle and heart differentiation processes were methylated during later stages of development. Taken together, our findings demonstrate the roles of COMPASS series complex core subunits Set1, Trx, and Trr in regulating histone methylation during heart development and, given their implication in congenital heart diseases, inform research on heart disease.
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Proteínas de Drosophila , Epigênese Genética , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Coração/crescimento & desenvolvimentoRESUMO
Drosophila is an important model for studying heart development and disease. Yet, single-cell transcriptomic data of its developing heart have not been performed. Here, we report single-cell profiling of the entire fly heart using â¼3000 Hand-GFP embryos collected at five consecutive developmental stages, ranging from bilateral migrating rows of cardiac progenitors to a fused heart tube. The data revealed six distinct cardiac cell types in the embryonic fly heart: cardioblasts, both Svp+ and Tin+ subtypes; and five types of pericardial cell (PC) that can be distinguished by four key transcription factors (Eve, Odd, Ct and Tin) and include the newly described end of the line PC. Notably, the embryonic fly heart combines transcriptional signatures of the mammalian first and second heart fields. Using unique markers for each heart cell type, we defined their number and location during heart development to build a comprehensive 3D cell map. These data provide a resource to track the expression of any gene in the developing fly heart, which can serve as a reference to study genetic perturbations and cardiac diseases.
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Drosophila melanogaster , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Coração/embriologia , Análise da Expressão Gênica de Célula Única , Linfonodos/citologia , Linfonodos/embriologia , Embrião não Mamífero , Desenvolvimento Embrionário , Biomarcadores , OrganogêneseRESUMO
Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. Here, we used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.
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The inference of Gene Regulatory Networks (GRNs) is one of the key challenges in systems biology. Leading algorithms utilize, in addition to gene expression, prior knowledge such as Transcription Factor (TF) DNA binding motifs or results of TF binding experiments. However, such prior knowledge is typically incomplete, therefore, integrating it with gene expression to infer GRNs remains difficult. To address this challenge, we introduce NetREX-CF-Regulatory Network Reconstruction using EXpression and Collaborative Filtering-a GRN reconstruction approach that brings together Collaborative Filtering to address the incompleteness of the prior knowledge and a biologically justified model of gene expression (sparse Network Component Analysis based model). We validated the NetREX-CF using Yeast data and then used it to construct the GRN for Drosophila Schneider 2 (S2) cells. To corroborate the GRN, we performed a large-scale RNA-Seq analysis followed by a high-throughput RNAi treatment against all 465 expressed TFs in the cell line. Our knockdown result has not only extensively validated the GRN we built, but also provides a benchmark that our community can use for evaluating GRNs. Finally, we demonstrate that NetREX-CF can infer GRNs using single-cell RNA-Seq, and outperforms other methods, by using previously published human data.
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Redes Reguladoras de Genes , Fatores de Transcrição , Humanos , Animais , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Biologia de Sistemas , Drosophila/genética , Saccharomyces cerevisiae/genética , Expressão GênicaRESUMO
SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection. Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1). Complementing transcriptomic data from the fly heart reveal that this interaction blocks the antagonistic MGA/MAX complex, which shifts the balance towards MYC/MAX and activates glycolysis-with similar findings in mouse cardiomyocytes. Further, the Nsp6-induced glycolysis disrupts cardiac mitochondrial function, known to increase reactive oxygen species (ROS) in heart failure; this could explain COVID-19-associated cardiac pathology. Inhibiting the glycolysis pathway by 2-deoxy-D-glucose (2DG) treatment attenuates the Nsp6-induced cardiac phenotype in flies and mice. These findings point to glycolysis as a potential pharmacological target for treating COVID-19-associated heart failure.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , COVID-19 , Proteínas de Drosophila/metabolismo , Insuficiência Cardíaca , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Desoxiglucose/metabolismo , Drosophila/metabolismo , Glicólise , Insuficiência Cardíaca/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , SARS-CoV-2RESUMO
Ran's GTPase-activating protein (RanGAP) is tethered to the nuclear envelope (NE) in multicellular organisms. We investigated the consequences of RanGAP localization in human tissue culture cells and Drosophila. In tissue culture cells, disruption of RanGAP1 NE localization surprisingly has neither obvious impacts on viability nor nucleocytoplasmic transport of a model substrate. In Drosophila, we identified a region within nucleoporin dmRanBP2 required for direct tethering of dmRanGAP to the NE. A dmRanBP2 mutant lacking this region shows no apparent growth defects during larval stages but arrests at the early pupal stage. A direct fusion of dmRanGAP to the dmRanBP2 mutant rescues this arrest, indicating that dmRanGAP recruitment to dmRanBP2 per se is necessary for the pupal ecdysis sequence. Our results indicate that while the NE localization of RanGAP is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, we find a requirement for this localization during pupal development.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Pupa/crescimento & desenvolvimento , Transporte Ativo do Núcleo Celular , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HCT116 , Humanos , Chaperonas Moleculares/genética , Membrana Nuclear/genética , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Pupa/genética , Pupa/metabolismoRESUMO
Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.
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Células-Tronco Adultas/metabolismo , Proteínas de Anfíbios/genética , Células Epiteliais/metabolismo , Intestinos/citologia , Larva/genética , Receptores dos Hormônios Tireóideos/genética , Hormônios Tireóideos/genética , Xenopus/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Proteínas de Anfíbios/classificação , Proteínas de Anfíbios/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Ontologia Genética , Redes Reguladoras de Genes , Intestinos/efeitos dos fármacos , Intestinos/crescimento & desenvolvimento , Larva/citologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Redes e Vias Metabólicas/genética , Metamorfose Biológica , Anotação de Sequência Molecular , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Receptores dos Hormônios Tireóideos/deficiência , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/farmacologia , Via de Sinalização Wnt/genética , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismoRESUMO
BACKGROUND: SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology. METHODS: We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction. RESULTS: We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells. CONCLUSIONS: Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.
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BACKGROUND: SARS-CoV-2 causes COVID-19 with a widely diverse disease profile that affects many different tissues. The mechanisms underlying its pathogenicity in host organisms remain unclear. Animal models for studying the pathogenicity of SARS-CoV-2 proteins are lacking. METHODS: Using bioinformatic analysis, we found that 90% of the virus-host interactions involve human proteins conserved in Drosophila. Therefore, we generated a series of transgenic fly lines for individual SARS-CoV-2 genes, and used the Gal4-UAS system to express these viral genes in Drosophila to study their pathogenicity. RESULTS: We found that the ubiquitous expression of Orf6, Nsp6 or Orf7a in Drosophila led to reduced viability and tissue defects, including reduced trachea branching as well as muscle deficits resulting in a "held-up" wing phenotype and poor climbing ability. Furthermore, muscles in these flies showed dramatically reduced mitochondria. Since Orf6 was found to interact with nucleopore proteins XPO1, we tested Selinexor, a drug that inhibits XPO1, and found that it could attenuate the Orf6-induced lethality and tissue-specific phenotypes observed in flies. CONCLUSIONS: Our study established Drosophila as a model for studying the function of SARS-CoV2 genes, identified Orf6 as a highly pathogenic protein in various tissues, and demonstrated the potential of Selinexor for inhibiting Orf6 toxicity using an in vivo animal model system.
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Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.
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Exodesoxirribonucleases/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares , Proteínas de Transporte Nucleocitoplasmático , Transporte Proteico , Proteínas de Ligação a RNA , Transcriptoma , Dedos de ZincoRESUMO
The maintenance of the intestinal epithelium is ensured by the controlled proliferation of intestinal stem cells (ISCs) and differentiation of their progeny into various cell types, including enterocytes (ECs) that both mediate nutrient absorption and provide a barrier against pathogens. The signals that regulate transition of proliferative ISCs into differentiated ECs are not fully understood. IRBIT is an evolutionarily conserved protein that regulates ribonucleotide reductase (RNR), an enzyme critical for the generation of DNA precursors. Here, we show that IRBIT expression in ISC progeny within the Drosophila midgut epithelium cells regulates their differentiation via suppression of RNR activity. Disruption of this IRBIT-RNR regulatory circuit causes a premature loss of intestinal tissue integrity. Furthermore, age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny. Collectively, our findings demonstrate a role of the IRBIT-RNR pathway in gut homeostasis.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Following publication of the original article [1], the authors reported the following errors.
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Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. To investigate Wolbachia-insect interactions we used the Wolbachia/Drosophila model that provides a genetically tractable system for studying host-pathogen interactions. We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship.
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Drosophila melanogaster/genética , Interações entre Hospedeiro e Microrganismos/genética , Wolbachia/genética , Animais , Culicidae , Drosophila/genética , Drosophila/microbiologia , Drosophila melanogaster/microbiologia , Genoma , Interações Hospedeiro-Patógeno/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Mosquitos Vetores , Interferência de RNA , Simbiose , Vírus/genética , Sequenciamento Completo do Genoma/métodosRESUMO
Gene regulatory networks (GRNs) describe regulatory relationships between transcription factors (TFs) and their target genes. Computational methods to infer GRNs typically combine evidence across different conditions to infer context-agnostic networks. We develop a method, Network Reprogramming using EXpression (NetREX), that constructs a context-specific GRN given context-specific expression data and a context-agnostic prior network. NetREX remodels the prior network to obtain the topology that provides the best explanation for expression data. Because NetREX utilizes prior network topology, we also develop PriorBoost, a method that evaluates a prior network in terms of its consistency with the expression data. We validate NetREX and PriorBoost using the "gold standard" E. coli GRN from the DREAM5 network inference challenge and apply them to construct sex-specific Drosophila GRNs. NetREX constructed sex-specific Drosophila GRNs that, on all applied measures, outperform networks obtained from other methods indicating that NetREX is an important milestone toward building more accurate GRNs.
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Reprogramação Celular/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Caracteres Sexuais , Animais , Escherichia coli/metabolismo , Feminino , Masculino , Reprodutibilidade dos TestesRESUMO
BACKGROUND: In animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation. RESULTS: We asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components. CONCLUSIONS: Our results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.
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Montagem e Desmontagem da Cromatina , Cromossomos de Insetos/genética , Mecanismo Genético de Compensação de Dose , Cromossomo X/genética , Animais , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Hemizigoto , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
DNA copy number variation is associated with many high phenotypic heterogeneity disorders. We systematically examined the impact of Drosophila melanogaster deletions on gene expression profiles to ask whether increased expression variability owing to reduced gene dose might underlie this phenotypic heterogeneity. Indeed, we found that one-dose genes have higher gene expression variability relative to two-dose genes. We then asked whether this increase in variability could be explained by intrinsic noise within cells due to stochastic biochemical events, or whether expression variability is due to extrinsic noise arising from more complex interactions. Our modeling showed that intrinsic gene expression noise averages at the organism level and thus cannot explain increased variation in one-dose gene expression. Interestingly, expression variability was related to the magnitude of expression compensation, suggesting that regulation, induced by gene dose reduction, is noisy. In a remarkable exception to this rule, the single X chromosome of males showed reduced expression variability, even compared with two-dose genes. Analysis of sex-transformed flies indicates that X expression variability is independent of the male differentiation program. Instead, we uncovered a correlation between occupancy of the chromatin-modifying protein encoded by males absent on the first (mof) and expression variability, linking noise suppression to the specialized X chromosome dosage compensation system. MOF occupancy on autosomes in both sexes also lowered transcriptional noise. Our results demonstrate that gene dose reduction can lead to heterogeneous responses, which are often noisy. This has implications for understanding gene network regulatory interactions and phenotypic heterogeneity. Additionally, chromatin modification appears to play a role in dampening transcriptional noise.
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Variações do Número de Cópias de DNA , Drosophila melanogaster/genética , Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Cromossomo X/genética , Animais , Cromatina/genética , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Feminino , Masculino , Processos EstocásticosRESUMO
Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.