RESUMEN
Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.
Asunto(s)
Sistemas CRISPR-Cas/genética , Factores de Transcripción Forkhead/metabolismo , Neoplasias/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismo , Animales , Autoinmunidad/inmunología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleosomas/inmunología , ARN Guía de Kinetoplastida/genética , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Factores de Transcripción/genéticaRESUMEN
T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor RORγt. Here, we identify REV-ERBα (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes RORγt function in Th17 cells. REV-ERBα binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of RORγt-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERBα expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células Th17/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Sitios Genéticos , Células HEK293 , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Ratones , Ratones Transgénicos , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , RNA-Seq , Elementos de Respuesta/genética , Células Th17/metabolismo , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
Communication between neighboring tissues plays a central role in guiding organ morphogenesis. During heart tube assembly, interactions with the adjacent endoderm control the medial movement of cardiomyocytes, a process referred to as cardiac fusion. However, the molecular underpinnings of this endodermal-myocardial relationship remain unclear. Here, we show an essential role for platelet-derived growth factor receptor alpha (Pdgfra) in directing cardiac fusion. Mutation of pdgfra disrupts heart tube assembly in both zebrafish and mouse. Timelapse analysis of individual cardiomyocyte trajectories reveals misdirected cells in zebrafish pdgfra mutants, suggesting that PDGF signaling steers cardiomyocytes toward the midline during cardiac fusion. Intriguingly, the ligand pdgfaa is expressed in the endoderm medial to the pdgfra-expressing myocardial precursors. Ectopic expression of pdgfaa interferes with cardiac fusion, consistent with an instructive role for PDGF signaling. Together, these data uncover a novel mechanism through which endodermal-myocardial communication can guide the cell movements that initiate cardiac morphogenesis.
Asunto(s)
Movimiento Celular , Corazón/embriología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Animales , Técnicas de Inactivación de Genes , Ratones , Morfogénesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Imagen de Lapso de Tiempo , Pez CebraRESUMEN
In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase.