RESUMEN
Overexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.
Asunto(s)
Drosophila/genética , Biblioteca de Genes , Técnicas Genéticas , Sistemas de Lectura Abierta , Animales , Código de Barras del ADN Taxonómico , Proteínas de Drosophila/genética , Epítopos/química , Plásmidos/metabolismo , TransgenesRESUMEN
Combinatorial interactions among transcription factors (TFs) play essential roles in generating gene expression specificity and diversity in metazoans. Using yeast 2-hybrid (Y2H) assays on nearly all sequence-specific Drosophila TFs, we identified 1,983 protein-protein interactions (PPIs), more than doubling the number of currently known PPIs among Drosophila TFs. For quality assessment, we validated a subset of our interactions using MITOMI and bimolecular fluorescence complementation assays. We combined our interactome with prior PPI data to generate an integrated Drosophila TF-TF binary interaction network. Our analysis of ChIP-seq data, integrating PPI and gene expression information, uncovered different modes by which interacting TFs are recruited to DNA. We further demonstrate the utility of our Drosophila interactome in shedding light on human TF-TF interactions. This study reveals how TFs interact to bind regulatory elements in vivo and serves as a resource of Drosophila TF-TF binary PPIs for understanding tissue-specific gene regulation.
Asunto(s)
Drosophila melanogaster/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , ADN/química , ADN/metabolismo , Regulación de la Expresión Génica , Microscopía Fluorescente , Mapas de Interacción de Proteínas/genética , Elementos Reguladores de la Transcripción , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods.We present a set of fly lines, called 'multicolor BiFC library', which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe two different binary interactions simultaneously and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Biblioteca de Genes , Mapeo de Interacción de Proteínas/métodos , Factores de Transcripción/genética , Animales , Animales Modificados Genéticamente , Color , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Fluorescencia , Regulación del Desarrollo de la Expresión Génica , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
Wnt ligands are lipid-modified, secreted glycoproteins that control multiple steps during embryogenesis and adult-tissue homeostasis. Little is known about the mechanisms underlying Wnt secretion. Recently, Wntless (Wls/Evi/Srt) was identified as a conserved multi-pass transmembrane protein whose function seems to be dedicated to promoting the release of Wnts. Here, we describe Wls accumulation in the Golgi apparatus of Wnt/Wingless (Wg)-producing cells in Drosophila, and show that this localization is essential for Wg secretion. Moreover, Wls localization and levels critically depend on retromer, a conserved protein complex that mediates endosome-to-Golgi protein trafficking in yeast. In the absence of the retromer components Dvps35 or Dvps26, but in presence of Wg, Wls is degraded and Wg secretion impaired. Our results indicate that Wg, clathrin-mediated endocytosis and retromer sustain a Wls traffic loop from the Golgi to the plasma membrane and back to the Golgi, thereby enabling Wls to direct Wnt secretion.