RESUMO
Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. Here, we developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. We observe extensive genome shuffling between these species with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.
Assuntos
Drosophila/genética , Evolução Molecular , Regulação da Expressão Gênica/genética , Genoma de Inseto/genética , Algoritmos , Animais , Cromossomos de Insetos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Drosophila/embriologia , Embrião não MamíferoRESUMO
Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. We propose that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Montagem e Desmontagem da Cromatina , Análise Citogenética , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Cromossomo X/genéticaRESUMO
Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.
Assuntos
Mecanismo Genético de Compensação de Dose/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Fatores de Transcrição/genética , Cromossomo X/genética , Animais , Animais Geneticamente Modificados , Pareamento de Bases , Western Blotting , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Imunoprecipitação , Masculino , Mutação/genética , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências de Repetição em Tandem/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Cromossomo X/metabolismoRESUMO
Endocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME.
Assuntos
Proteínas de Drosophila/genética , Endocitose/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Rodopsina/genética , Proteínas rab5 de Ligação ao GTP/genética , Absorciometria de Fóton , Animais , Membrana Celular/genética , Clatrina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismoRESUMO
During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Animais , Proliferação de Células , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Microscopia Confocal , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
During sensory transduction, Drosophila photoreceptors experience substantial increases in intracellular Ca(2+) levels ([Ca(2+)](i)). Nevertheless in a number of mutants associated with excessive Ca(2+) influx through transient receptor potential (TRP) channels, Drosophila photoreceptors undergo loss of normal cellular structure manifest as a retinal degeneration. However, the molecular mechanisms that underpin this degeneration process remain unclear. The authors previously isolated a mutant, su(40), that is able to suppress the retinal degeneration seen in photoreceptors from loss-of-function alleles of rdgA that are known to have constitutively active TRP channels. Here the authors report the genetic mapping of su(40) as well the isolation of additional alleles of su(40). Studies of su(40) as well as these new alleles should facilitate the understanding of the mechanisms by which excessive Ca(2+) influx results in retinal degeneration.
Assuntos
Proteínas de Drosophila/genética , Mutação/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Degeneração Retiniana , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Modelos Animais de Doenças , Drosophila melanogaster , Proteínas do Olho/genética , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Proteínas de Membrana/genética , Mutagênese/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosfolipase C beta/genética , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Previously, we demonstrated that IL-1ß was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1ß requires the activation of MAP kinase pathways as a consequence of an IL-1ß receptor stimulation. Moreover, we have observed that IL-1ß regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-κB partially mediated by p38 MAPK.