RESUMEN
Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Sistemas CRISPR-Cas , Calcio/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Mecanotransducción Celular/fisiología , Oncogenes/genética , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.
Asunto(s)
Sitios de Unión , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Nucleosomas/metabolismo , Secuencia de Bases , Línea Celular , Cromatina , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Unión Proteica , Dominios ProteicosRESUMEN
The promiscuity of G-protein-coupled receptors (GPCRs) has broad implications in disease, pharmacology and biosensing. Promiscuity is a particularly crucial consideration for protein engineering, where the ability to modulate and model promiscuity is essential for developing desirable proteins. Here, we present methodologies for (i) modifying GPCR promiscuity using directed evolution and (ii) predicting receptor response and identifying important peptide features using quantitative structure-activity relationship models and grouping-exhaustive feature selection. We apply these methodologies to the yeast pheromone receptor Ste2 and its native ligand α-factor. Using directed evolution, we created Ste2 mutants with altered specificity toward a library of α-factor variants. We then used the Vectors of Hydrophobic, Steric, and Electronic properties and partial least squares regression to characterize receptor-ligand interactions, identify important ligand positions and properties, and predict receptor response to novel ligands. Together, directed evolution and computational analysis enable the control and evaluation of GPCR promiscuity. These approaches should be broadly useful for the study and engineering of GPCRs and other protein-small molecule interactions.