RESUMEN
How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.
Asunto(s)
Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Interleucina-1alfa/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Sitios de Unión , Células Cultivadas , Quimiocinas/metabolismo , Cromatina/química , Cromatina/genética , Células HeLa , Humanos , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Signals and posttranslational modifications regulating the decapping step in mRNA degradation pathways are poorly defined. In this study we reveal the importance of K63-linked ubiquitylation for the assembly of decapping factors, P-body formation, and constitutive decay of instable mRNAs encoding mediators of inflammation by various experimental approaches. K63-branched ubiquitin chains also regulate IL-1-inducible phosphorylation of the P-body component DCP1a. The E3 ligase TRAF6 binds to DCP1a and indirectly regulates DCP1a phosphorylation, expression of decapping factors, and gene-specific mRNA decay. Mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs the interaction with the mRNA decay factors DCP2, EDC4, and XRN1, but not EDC3, thus remodeling P-body architecture. The usage of ubiquitin chains for the proper assembly and function of the decay-competent mammalian decapping complex suggests an additional layer of control to allow a coordinated function of decapping activities and mRNA metabolism in higher eukaryotes.
Asunto(s)
Endorribonucleasas/metabolismo , Lisina/metabolismo , Caperuzas de ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Transactivadores/metabolismo , Ubiquitinación , Animales , Línea Celular Tumoral , Endorribonucleasas/genética , Exorribonucleasas/metabolismo , Células HEK293 , Humanos , Interleucina-1alfa/farmacología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/metabolismo , Caperuzas de ARN/genética , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , Receptores de Interleucina-1/agonistas , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factores de Tiempo , Transactivadores/genética , Transfección , Ubiquitinación/efectos de los fármacosRESUMEN
The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells.
RESUMEN
Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.
Asunto(s)
Estrés del Retículo Endoplásmico , SARS-CoV-2/fisiología , Replicación Viral/fisiología , Animales , Autofagia/efectos de los fármacos , Bronquios/patología , COVID-19/patología , COVID-19/virología , Diferenciación Celular/efectos de los fármacos , Extractos Celulares , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Coronavirus Humano 229E/fisiología , Regulación hacia Abajo/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Proteínas de Choque Térmico/metabolismo , Humanos , Macrólidos/farmacología , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Biosíntesis de Proteínas/efectos de los fármacos , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
TAB1 was defined as a regulatory subunit of the protein kinase TAK1, which functions upstream in the pathways activated by interleukin (IL)-1, tumor necrosis factor (TNF), toll-like receptors (TLRs) and stressors. However, TAB1 also functions in the p38 MAPK pathway downstream of TAK1. We identified amino acids (aa) 452/453 and 456/457 of TAB1 as novel sites phosphorylated by TAK1 as well as by p38 MAPK in intact cells as well as in vitro. Serines 452/453 and 456/457 were phosphorylated upon phosphatase blockade by calyculin A, or in response to IL-1 or translational stressors such as anisomycin and sorbitol. Deletion or phospho-mimetic mutations of aa 452-457 of TAB1 retain TAB1 and p38 MAPK in the cytoplasm. The TAB1 mutant lacking aa 452-457 decreases TAB1-dependent phosphorylation of p38 MAPK. It also enhances TAB1-dependent CCL5 secretion in response to IL-1 and increases activity of a post-transcriptional reporter gene, which contains the CCL5 3' untranslated region. These data suggest a complex role of aa 452-457 of TAB1 in controlling p38 MAPK activity and subcellular localization and implicate these residues in TAK1- or p38 MAPK-dependent post-transcriptional control of gene expression.