RESUMEN
Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.
Asunto(s)
Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Células Madre Embrionarias , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Ratones , Mutación/genética , Dominios Proteicos , Estabilidad Proteica , Proteolisis , ARN Polimerasa II/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers--SRR18, SRR107, and SRR111--that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus(129) × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.
Asunto(s)
Diferenciación Celular , Cromatina/metabolismo , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Células Cultivadas , Ratones , Familia de Multigenes/genética , Placa Neural/citología , Regiones Promotoras Genéticas/genética , Eliminación de Secuencia/genéticaRESUMEN
Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Neuronas , Organoides , Fosfohidrolasa PTEN , Humanos , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Organoides/metabolismo , Neuronas/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Mutación/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Transducción de Señal , Proliferación Celular , Proteína Reguladora Asociada a mTOR/metabolismo , Proteína Reguladora Asociada a mTOR/genética , FenotipoRESUMEN
Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels, it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression . Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long non-coding RNAs including enhancer RNA (eRNA). Here, we describe nuclear RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long non-coding RNAs.
Asunto(s)
Análisis de Secuencia de ARN , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Poliadenilación , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Nuclear/genética , TranscriptomaRESUMEN
Cooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naive pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. We show that, as embryonic stem cells (ESCs) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naive pluripotent state and initiation of ESC differentiation.
Asunto(s)
Ciclo Celular , Núcleo Celular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transporte Activo de Núcleo Celular , Animales , Diferenciación Celular , Regulación hacia Abajo , Activación Enzimática , Carioferinas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Células Madre Embrionarias de Ratones , Proteína Homeótica Nanog/metabolismo , Señales de Exportación Nuclear , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Proteína Exportina 1RESUMEN
Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression. Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long noncoding RNAs. Here, we describe nuclear-RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long noncoding RNAs.
Asunto(s)
Perfilación de la Expresión Génica/métodos , ARN Nuclear/genética , ARN Nuclear/aislamiento & purificación , Análisis de Secuencia de ARN/métodos , Transcriptoma , Animales , Fraccionamiento Celular/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Poliadenilación , ARN Largo no Codificante/genética , ARN Largo no Codificante/aislamiento & purificaciónRESUMEN
Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells.
RESUMEN
Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.