RESUMEN
The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Regeneración , Animales , Proliferación Celular/efectos de los fármacos , Corazón/fisiología , Humanos , Células Madre Pluripotentes Inducidas , Regeneración Hepática/efectos de los fármacos , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Organoides/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Regeneración/genética , Retina/fisiología , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/genética , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.
Asunto(s)
Metilación de ADN/genética , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Mutación/genética , Síndrome de Rett/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG/deficiencia , Ratones , Datos de Secuencia Molecular , Neuronas/metabolismoRESUMEN
Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that, in neurons, regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MeCP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3',5'-cyclic AMP (cAMP), indicating that MeCP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MeCP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MeCP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MeCP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MeCP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MeCP2 at T308 regulates the interaction of MeCP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MeCP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MeCP2 with the NCoR complex.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Treonina/metabolismo , Animales , Células Cultivadas , Humanos , Proteína 2 de Unión a Metil-CpG/química , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Mutación , Neuronas/metabolismo , Fosforilación , Síndrome de Rett/genética , Transcripción GenéticaRESUMEN
Cardiomyocyte proliferation and dedifferentiation have fueled the field of regenerative cardiology in recent years, whereas the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterized by the restoration of function lost during dedifferentiation. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: transient dedifferentiation and redifferentiation. Here we survey the temporal transcriptomic and proteomic landscape of dedifferentiation-redifferentiation in adult mouse hearts and reveal that well-characterized dedifferentiation features largely return to normal, although elements of residual dedifferentiation remain, even after the contractile function is restored. These hearts appear rejuvenated and show robust resistance to ischemic injury, even 5 months after redifferentiation initiation. Cardiomyocyte redifferentiation is driven by negative feedback signaling and requires LATS1/2 Hippo pathway activity. Our data reveal the importance of cardiomyocyte redifferentiation in functional restoration during regeneration but also protection against future insult, in what could lead to a potential prophylactic treatment against ischemic heart disease for at-risk patients.
RESUMEN
The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP's potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and nonneuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington's Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP's transcriptional activity can partially rescue the HD phenotype.
Asunto(s)
Ectodermo , Enfermedad de Huntington , Proteínas Señalizadoras YAP , Proteínas de Ciclo Celular/metabolismo , Ectodermo/metabolismo , Humanos , Neurogénesis , Neurulación , Transducción de Señal/genética , Proteínas Señalizadoras YAP/genéticaRESUMEN
Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases-the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proliferación Celular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Células Ependimogliales/citología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células HEK293 , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Although a hair bundle is normally deflected by mechanical stimuli, we found that irradiation of a hair cell from the bullfrog's sacculus with ultraviolet light causes rapid motion of the hair bundle toward its tall edge. This movement is associated with opening of mechanotransduction channels and disappears when tip links are disrupted. We localized the absorptive element responsible for the motion to the region directly below the hair bundle and measured an action spectrum similar to the absorption spectra of mitochondrial constituents. Temperature measurements revealed heating around the site of absorption; direct heating of the hair bundle confirmed that the response to light is mediated through heat. Although mechanical offsets of the hair bundle revealed that heat softens gating springs, it also acts directly to open transduction channels. This study identifies an unconventional method of hair-cell stimulation and clarifies the previously unexplained sensitivity of auditory organs to thermal stimulation.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Mecanotransducción Celular , Rayos Ultravioleta , Animales , Femenino , Masculino , Estimulación Física , Rana catesbeiana , TemperaturaRESUMEN
Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.