RESUMEN
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.
Asunto(s)
Citoesqueleto/genética , Proteínas Activadoras de GTPasa/genética , Integrinas/genética , Mecanotransducción Celular/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína de Unión al GTP rac1/genética , Animales , Células COS , Adhesión Celular , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Biología Computacional , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Perros , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Integrinas/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Pan troglodytes , Dominios Proteicos , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/clasificación , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
R-loops are DNA-RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states1-8. How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref. 9), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs.
Asunto(s)
Proteínas de Ciclo Celular/genética , Islas de CpG/genética , Oxigenasas de Función Mixta/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Ciclo Celular/genética , Línea Celular , Cromatina/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Células HEK293 , Humanos , Ratones , Unión Proteica/genética , ARN Largo no Codificante/genética , Ribonucleasa H/genética , Transcripción Genética/genéticaRESUMEN
Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo-endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration.