Drosophila Genome-Wide RNAi Screen Identifies Multiple Regulators of HIF–Dependent Transcription in Hypoxia

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke

Epigenetics: New Questions on the Response to Hypoxia

Reduction in oxygen levels below normal concentrations plays important roles in different normal and pathological conditions, such as development, tumorigenesis, chronic kidney disease and stroke. Organisms exposed to hypoxia trigger changes at both cellular and systemic levels to recover oxygen homeostasis. Most of these processes are mediated by Hypoxia Inducible Factors, HIFs, a family of transcription factors that directly induce the expression of several hundred genes in mammalian cells. Although different aspects of HIF regulation are well known, it is still unclear by which precise mechanism HIFs activate transcription of their target genes. Concomitantly, hypoxia provokes a dramatic decrease of general transcription that seems to rely in part on epigenetic changes through a poorly understood mechanism. In this review we discuss the current knowledge on chromatin changes involved in HIF dependent gene activation, as well as on other epigenetic changes, not necessarily linked to HIF that take place under hypoxic conditions

The TIP60 complex is a conserved coactivator of HIF1A

Hypoxia-inducible factors (HIFs) are critical regulators of the cellular response to hypoxia. Despite their established roles in normal physiology and numerous pathologies, the molecular mechanisms by which they control gene expression remain poorly understood. We report here a conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells. TIP60 (KAT5) is required for HIF1-dependent gene expression in fly cells and embryos and colorectal cancer cells. HIF1A interacts with and recruits TIP60 to chromatin. TIP60 is dispensable for HIF1A association with its target genes but is required for HIF1A-dependent chromatin modification and RNA polymerase II activation in hypoxia. In human cells, global analysis of HIF1A-dependent gene activity reveals that most HIF1A targets require either TIP60, the CDK8-Mediator complex, or both as coactivators for full expression in hypoxia. Thus, HIF1A employs functionally diverse cofactors to regulate different subsets of genes within its transcriptional program.Fil: Perez Perri, Joel Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dengler, Verónica L.. Howard Hughes Medical Institute; Estados UnidosFil: Audetat, Audrey. Howard Hughes Medical Institute; Estados UnidosFil: Pandey, Ahwan. Howard Hughes Medical Institute; Estados UnidosFil: Bonner, Elyzabeth A.. State University Of Colorado Boulder; Estados UnidosFil: Urh, Marjeta. Promega Corporation; Estados UnidosFil: Mendez, Jacqui. Promega Corporation; Estados UnidosFil: Daniels, Danette L.. Promega Corporation; Estados UnidosFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Galbraith, Matthew D.. Howard Hughes Medical Institute; Estados UnidosFil: Espinosa, Joaquín M.. Howard Hughes Medical Institute; Estados Unido

The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Recent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs.<br/

The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Recent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs

Zonda is a novel early component of the autophagy pathway in Drosophila

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation.Fil: Melani, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Valko, Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Romero, Nuria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Aguilera, Milton Osmar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Acevedo, Julieta María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bhujabal, Zambarlal. The Arctic University of Norway; NoruegaFil: Perez Perri, Joel Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de la Riva Carrasco, Rocío Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Sorianello, Eleonora Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: D'Alessio, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Juhász, Gabor. Hungary Institute of Genetics; HungríaFil: Johansen, Terje. The Arctic University Of Norway; NoruegaFil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin