RESUMEN
MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at â¼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.
Asunto(s)
Carcinogénesis/metabolismo , Cromatina/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anisotropía , Sitios de Unión/genética , Carcinogénesis/genética , Cromatina/química , Cromatina/genética , Polarización de Fluorescencia , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.
Asunto(s)
Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/fisiología , Pérdida de Heterocigocidad , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Adolescente , Adulto , Alelos , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Genes p53 , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Masculino , Mutación , ARN Interferente Pequeño/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current ('pump current'). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to -80â¯mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.
Asunto(s)
Hemiplejía/diagnóstico , Hemiplejía/fisiopatología , Potenciales de la Membrana/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Adulto , Animales , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Técnicas de Cocultivo , Femenino , Hemiplejía/genética , Humanos , Lactante , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Ratones , Adulto JovenRESUMEN
Tuberous sclerosis complex (TSC) is a genetic disease with severe neurologic and psychiatric manifestations including epilepsy, developmental delay, and autism. Despite much progress in defining abnormal signaling pathways including the contribution of increased mTORC1 signaling, specific abnormalities that underlie the severe neurologic features in TSC remain poorly understood. We hypothesized that epilepsy and autism in TSC result from abnormalities of γ-aminobutyric acidergic (GABAergic) interneurons. To test this hypothesis, we generated conditional knockout mice with selective deletion of the Tsc1 gene in GABAergic interneuron progenitor cells. These interneuron-specific Tsc1 conditional knockout (CKO) mice have impaired growth and decreased survival. Cortical and hippocampal GABAergic interneurons of CKO mice are enlarged and show increased mTORC1 signaling. Total numbers of GABAergic cells are reduced in the cortex with differential reduction of specific GABAergic subtypes. Ectopic clusters of cells with increased mTORC1 signaling are also seen suggesting impaired interneuron migration. The functional consequences of these cellular changes are evident in the decreased seizure threshold on exposure to the proconvulsant flurothyl. These findings support an important role for the Tsc1 gene during GABAergic interneuron development, function, and possibly migration.