title: Integrating phosphoproteome and transcriptome reveals new determinants of macrophage multinucleation.
abstract: Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.
authors.affiliation: From the ‡Integrative Genomics and Medicine, MRC Clinical Sciences Centre, Imperial College London, UK;
authors.forename: Maxime
authors.initials: M
authors.lastname: Rotival
authors.affiliation: §Centre for Complement and Inflammation Research (CCIR), Imperial College London, UK;
authors.forename: Jeong-Hun
authors.initials: JH
authors.lastname: Ko
authors.affiliation: From the ‡Integrative Genomics and Medicine, MRC Clinical Sciences Centre, Imperial College London, UK;
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Srivastava
authors.affiliation: §Centre for Complement and Inflammation Research (CCIR), Imperial College London, UK;
authors.forename: Audrey
authors.initials: A
authors.lastname: Kerloc'h
authors.affiliation: ‖Biological Mass Spectrometry and Proteomics Laboratory, MRC Clinical Sciences Centre, Imperial College London, UK;
authors.forename: Alex
authors.initials: A
authors.lastname: Montoya
authors.affiliation: ¶William Harvey Research Institute, Queen Mary University of London, UK;
authors.forename: Claudio
authors.initials: C
authors.lastname: Mauro
authors.affiliation: ‖Biological Mass Spectrometry and Proteomics Laboratory, MRC Clinical Sciences Centre, Imperial College London, UK;
authors.forename: Peter
authors.initials: P
authors.lastname: Faull
authors.affiliation: **Integrative Cell Signaling and Proteomics, Barts Cancer Institute, Queen Mary University of London, UK.
authors.forename: Pedro R
authors.initials: PR
authors.lastname: Cutillas
authors.affiliation: From the ‡Integrative Genomics and Medicine, MRC Clinical Sciences Centre, Imperial College London, UK; Jacquesb@imperial.ac.uk enrico.petretto@duke-nus.edu.sg.
authors.forename: Enrico
authors.initials: E
authors.lastname: Petretto
authors.affiliation: §Centre for Complement and Inflammation Research (CCIR), Imperial College London, UK; Jacquesb@imperial.ac.uk enrico.petretto@duke-nus.edu.sg.
authors.forename: Jacques
authors.initials: J
authors.lastname: Behmoaras
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008264:Macrophages / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D011921:Rats, Inbred WKY; D017423:Sequence Analysis, RNA / Q000379:methods; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C583985:LRRFIP1 protein, rat; D009687:Nuclear Proteins; D020543:Proteome; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619382:Dnmt1 protein, rat; C578929:Smarca4 protein, rat; D004265:DNA Helicases