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1.
Cancer Sci ; 111(1): 148-159, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31733123

RESUMEN

The majority of breast cancers are primarily hormone-sensitive and can be managed by endocrine therapy, although therapy-resistant or hormone-refractory cancers need alternative treatments. Recently, increasing attention is being paid to RNA-binding proteins (RBP) in cancer pathophysiology. The precise role of RBP in breast cancer, however, remains to be clarified. We herein show that an RBP non-POU domain-containing octamer binding (NONO) plays a critical role in the pathophysiology of breast cancers regardless of their hormone dependency. Clinicopathological and immunohistochemical study of 127 breast cancer cases showed that NONO is a significant independent prognostic factor for breast cancer patients. Notably, siRNA-mediated NONO knockdown substantially repressed the proliferation of both hormone-sensitive MCF-7 and hormone-refractory MB-MDA-231 breast cancer cells. Integrative analysis combined with expression microarray and RIP-sequencing (RNA immunoprecipitation-sequencing) showed that NONO post-transcriptionally regulates the expression of cell proliferation-related genes by binding to their mRNAs, as exemplified by S-phase-associated kinase 2 and E2F transcription factor 8. Overall, these results suggest that NONO is a key regulator for breast cancer proliferation through the pre-mRNA splicing of cell proliferation-related genes and could be a potential new diagnostic and therapeutic target for advanced disease.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inmunoprecipitación/métodos , Células MCF-7 , ARN Mensajero/genética
2.
Cancer Sci ; 111(1): 239-252, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31729096

RESUMEN

Hypoxia-inducible factor 1 (HIF-1) is a critical heterodimeric transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) has been reported to function as a deubiquitinating enzyme for the stabilization of its α subunit (HIF-1α). In the present study, we showed that UCHL1 inhibition can be an effective therapeutic strategy against HIF-1-dependent tumor malignancy. In 2D monolayer culture, a UCHL1 inhibitor suppressed HIF activity and decreased the transcription of HIF downstream genes by inhibiting the UCHL1-mediated accumulation of HIF-1α. Phenotypically, UCHL1 inhibition remarkably blocked cell migration. In 3D spheroid culture models, ectopic expression of UCHL1 significantly upregulated malignancy-related factors such as solidity, volume, as well as viable cell number in an HIF-1α-dependent manner. Conversely, inhibition of the UCHL1-HIF-1 pathway downregulated these malignancy-related factors and also abolished UCHL1-mediated cell proliferation and invasiveness. Finally, inhibition of UCHL1 promoted HIF-1α degradation and lowered the expression of HIF-1 target genes in the 3D model, as also observed in 2D monolayer culture. Our research indicates that the UCHL1-HIF-1 pathway plays a crucial role in tumor malignancy, making it a promising therapeutic target for cancer chemotherapy.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Esferoides Celulares/patología , Ubiquitina Tiolesterasa/genética , Ubiquitinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Regulación hacia Arriba/genética
5.
Biochim Biophys Acta Gene Regul Mech ; 1862(10): 194438, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31634637

RESUMEN

Calpain 2 (CAPN2) is a Ca2+-dependent cysteine-protease that is involved in different cellular processes. Despite its important role, little is known about how CAPN2 expression is regulated. This study addressed the potential regulation of CAPN2 by microRNAs (miRNAs) in human endothelial cells. Two miRNAs were found to regulate CAPN2 expression by two distinct mechanisms, one direct and the other indirect. MiR-223 directly targeted CAPN2 by binding to the CAPN2 3'-untranslated region. Mir-223 overexpression decreased CAPN2 protein levels in cultured cells and in mice miR-223 antagonism led to an increase in CAPN2 protein in lung tissue. MiR-145 overexpression also decreased CAPN2 expression but did not affect a CAPN2 luciferase construct, indicating that the effect was indirect. MiR-145 targets histone deacetylase (HDAC) 2, and HDAC inhibition transcriptionally regulated CAPN2 expression by hyperacetylation of the promoter of CAPN2 gene and a subsequent decrease in polymerase 2 binding. Indeed, down regulation of HDAC2 by miR-145 not only decreased CAPN2 protein expression and calpain activity, but also protected paxillin against calpain-dependent degradation. Thus, protein levels of CAPN2 are regulated by miR-223, acting directly on the 3'-untranslated region as well as by miR-145, which acts via an increase in HDAC2. ENZYMES: Calpain 2 (EC 3.4.22.53), histone deacetylase 2 (EC 3.5.1.98).


Asunto(s)
Calpaína/genética , Células Endoteliales/metabolismo , MicroARNs/genética , Regiones no Traducidas 3' , Animales , Línea Celular , Regulación de la Expresión Génica/genética , Histona Desacetilasa 2/genética , Humanos , Ratones , Regiones Promotoras Genéticas
6.
Biochim Biophys Acta Gene Regul Mech ; 1862(10): 194442, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31634638

RESUMEN

MyoD is a determining transcription factor involved in myogenic cell differentiation. Post translational modifications of MyoD, including phosphorylation and acetylation, can regulate its transcription activity. Inhibition of protein arginine methyltransferase 1 (PRMT1) leads to insufficient muscle differentiation. However, little is known about arginine methylation in regulating MyoD activity. Here, we demonstrated that MyoD interacts with PRMT1 via its bHLH domain. MyoD could be methylated by PRMT1 at R121. Moreover, R111 and R121 of MyoD are responsible for MyoD-mediated myogenin gene transcription in C2C12 cells. PRMT1 promotes MyoD-mediated myogenin expression, for which the enzymatic activity of PRMT1 is needed. The arginine methylation of MyoD by PRMT1 enhances its DNA binding activity and transactivation. Our data help to further clarify the molecular mechanism of PRMT1 in regulating muscle cell differentiation and provide a new therapeutic target for diseases caused by the abnormal differentiation of muscle cells.


Asunto(s)
Proteína MioD/genética , Miogenina/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Represoras/genética , Transcripción Genética , Arginina/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/genética , Humanos , Metilación , Desarrollo de Músculos/genética , Procesamiento Proteico-Postraduccional/genética
7.
Nat Commun ; 10(1): 4784, 2019 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-31636266

RESUMEN

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, and protein manufacturing. The most successful approaches to date are based on modulating mRNA synthesis via an inducible coupling to transcriptional effectors. Here we present a biological programming structure that leverages a system of engineered transcription factors and complementary genetic architectures. We use a modular design strategy to create 27 non-natural and non-synonymous transcription factors using the lactose repressor topology as a guide. To direct systems of engineered transcription factors we employ parallel and series genetic (DNA) architectures and confer fundamental and combinatorial logical control over gene expression. Here we achieve AND, OR, NOT, and NOR logical controls in addition to two non-canonical half-AND operations. The basic logical operations and corresponding parallel and series genetic architectures represent the building blocks for subsequent combinatorial programs, which display both digital and analog performance.


Asunto(s)
Regulación de la Expresión Génica/genética , Ingeniería Metabólica/métodos , Factores de Transcripción/genética , Activación Transcripcional/genética , ADN , Redes Reguladoras de Genes , Ingeniería Genética/métodos , Vectores Genéticos , Biología Sintética , Transcripción Genética
8.
Nucleic Acids Res ; 47(19): 10072-10085, 2019 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-31665742

RESUMEN

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , ADN Mitocondrial/genética , Regulación de la Expresión Génica/genética , Animales , Islas de CpG/genética , Humanos , Mitocondrias/genética
9.
Genes Dev ; 33(21-22): 1591-1612, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31601616

RESUMEN

Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. Here, we developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. We observe extensive genome shuffling between these species with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.


Asunto(s)
Drosophila/genética , Evolución Molecular , Regulación de la Expresión Génica/genética , Genoma de los Insectos/genética , Algoritmos , Animales , Cromosomas de Insectos/genética , Secuencia Conservada , Compensación de Dosificación (Genética) , Drosophila/embriología , Embrión no Mamífero
10.
PLoS Genet ; 15(10): e1008357, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31609978

RESUMEN

Nonsyndromic orofacial cleft (NSOFC) is a severe birth defect that occurs early in embryonic development and includes the subtypes cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP). Given a lack of specific genetic factor analysis for CPO and CLO, the present study aimed to dissect the landscape of genetic factors underlying the pathogenesis of these two subtypes using 6,986 cases and 10,165 controls. By combining a genome-wide association study (GWAS) for specific subtypes of CPO and CLO, as well as functional gene network and ontology pathway analysis, we identified 18 genes/loci that surpassed genome-wide significance (P < 5 × 10-8) responsible for NSOFC, including nine for CPO, seven for CLO, two for both conditions and four that contribute to the CLP subtype. Among these 18 genes/loci, 14 are novel and identified in this study and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. Interestingly, we observed an opposite effect of the genetic variants in the IRF6 gene for CPO and CLO. Moreover, the gene expression dosage effect of IRF6 with two different alleles at the same single-nucleotide polymorphism (SNP) plays important roles in driving CPO or CLO. In addition, PAX9 is a key TF for CPO. Our findings define subtypes of NSOFC using genetic factors and their functional ontologies and provide a clue to improve their diagnosis and treatment in the future.


Asunto(s)
Encéfalo/anomalías , Labio Leporino/genética , Fisura del Paladar/genética , Factores Reguladores del Interferón/genética , Factor de Transcripción PAX9/genética , Alelos , Encéfalo/fisiopatología , Labio Leporino/fisiopatología , Fisura del Paladar/fisiopatología , Dosificación de Gen/genética , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Polimorfismo de Nucleótido Simple/genética
11.
Genes Dev ; 33(19-20): 1346-1354, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31575678

RESUMEN

The homologous recombination (HR) machinery plays multiple roles in genome maintenance. Best studied in the context of DNA double-stranded break (DSB) repair, recombination enzymes can cleave, pair, and unwind DNA molecules, and collaborate with regulatory proteins to execute multiple DNA processing steps before generating specific repair products. HR proteins also help to cope with problems arising from DNA replication, modulating impaired replication forks or filling DNA gaps. Given these important roles, it is not surprising that each HR step is subject to complex regulation to adjust repair efficiency and outcomes as well as to limit toxic intermediates. Recent studies have revealed intricate regulation of all steps of HR by the protein modifier SUMO, which has been increasingly recognized for its broad influence in nuclear functions. This review aims to connect established roles of SUMO with its newly identified effects on recombinational repair and stimulate further thought on many unanswered questions.


Asunto(s)
Recombinación Homóloga/genética , Proteína SUMO-1/metabolismo , Animales , Regulación de la Expresión Génica/genética , Humanos , Recombinasa Rad51/metabolismo , Sumoilación
12.
Nat Commun ; 10(1): 4481, 2019 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-31578371

RESUMEN

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.


Asunto(s)
Medios de Cultivo Condicionados/farmacología , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Luminiscentes/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Trimetoprim/farmacología , Animales , Antiinfecciosos/farmacología , Citometría de Flujo , Regulación de la Expresión Génica/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Ratones , Microscopía Confocal
13.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31570894

RESUMEN

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Asunto(s)
Linfocitos B/inmunología , Cromatina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Asesinas Naturales/inmunología , Elementos de Respuesta/genética , Linfocitos T/inmunología , Desequilibrio Alélico , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/inmunología , Epigénesis Genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-2/farmacología , Interleucina-4/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Polisacáridos/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Transcriptoma
14.
Nat Commun ; 10(1): 4171, 2019 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-31519904

RESUMEN

The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.


Asunto(s)
Proteína 1 Similar a ELAV/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , Neoplasias/complicaciones , Animales , Línea Celular , Línea Celular Tumoral , Estudios Transversales , Proteína 1 Similar a ELAV/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados
15.
Nat Commun ; 10(1): 4181, 2019 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-31519907

RESUMEN

The stability and quality of metazoan mRNAs are under microRNA (miRNA)-mediated and nonsense-mediated control. Although UPF1, a core mediator of nonsense-mediated mRNA decay (NMD), mediates the decay of target mRNA in a 3'UTR-length-dependent manner, the detailed mechanism remains unclear. Here, we suggest that 3'UTR-length-dependent mRNA decay is not mediated by nonsense mRNAs but rather by miRNAs that downregulate target mRNAs via Ago-associated UPF1/SMG7. Global analyses of mRNAs in response to UPF1 RNA interference in miRNA-deficient cells reveal that 3'UTR-length-dependent mRNA decay by UPF1 requires canonical miRNA targeting. The destabilization of miRNA targets is accomplished by the combination of Ago2 and UPF1/SMG7, which may recruit the CCR4-NOT deadenylase complex. Indeed, loss of the SMG7-deadenylase complex interaction increases the levels of transcripts regulated by UPF1-SMG7. This UPF1/SMG7-dependent miRNA-mediated mRNA decay pathway may enable miRNA targeting to become more predictable and expand the miRNA-mRNA regulatory network.


Asunto(s)
Proteínas Portadoras/metabolismo , Biología Computacional/métodos , MicroARNs/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN/fisiología , Transactivadores/metabolismo , Regiones no Traducidas 3'/genética , Animales , Western Blotting , Proteínas Portadoras/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Ratones , MicroARNs/genética , ARN Helicasas/genética , Interferencia de ARN/fisiología , Estabilidad del ARN/genética , Transactivadores/genética
16.
PLoS Comput Biol ; 15(9): e1007329, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31509524

RESUMEN

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism's complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors.


Asunto(s)
Biología Computacional/métodos , Código de Histonas/genética , Modelos Estadísticos , Nucleosomas/genética , Plasmodium falciparum/genética , Eritrocitos/parasitología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Genes Protozoarios/genética , Humanos , Estadios del Ciclo de Vida/genética , Aprendizaje Automático , Malaria Falciparum , Modelos Biológicos , Plasmodium falciparum/citología , Plasmodium falciparum/patogenicidad
17.
Nucleic Acids Res ; 47(19): 10439-10451, 2019 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-31511890

RESUMEN

One challenge in engineering organisms is taking responsibility for their behavior over many generations. Spontaneous mutations arising before or during use can impact heterologous genetic functions, disrupt system integration, or change organism phenotype. Here, we propose restructuring the genetic code itself such that point mutations in protein-coding sequences are selected against. Synthetic genetic systems so-encoded should fail more safely in response to most spontaneous mutations. We designed fail-safe codes and simulated their expected effects on the evolution of so-encoded proteins. We predict fail-safe codes supporting expression of 20 or 15 amino acids could slow protein evolution to ∼30% or 0% the rate of standard-encoded proteins, respectively. We also designed quadruplet-codon codes that should ensure all single point mutations in protein-coding sequences are selected against while maintaining expression of 20 or more amino acids. We demonstrate experimentally that a reduced set of 21 tRNAs is capable of expressing a protein encoded by only 20 sense codons, whereas a standard 64-codon encoding is not expressed. Our work suggests that biological systems using rationally depleted but otherwise natural translation systems should evolve more slowly and that such hypoevolvable organisms may be less likely to invade new niches or outcompete native populations.


Asunto(s)
Biología Computacional , Evolución Molecular , Código Genético/genética , Modelos Teóricos , Aminoácidos/genética , Regulación de la Expresión Génica/genética , Sistemas de Lectura Abierta/genética , Mutación Puntual/genética , Biosíntesis de Proteínas/genética , ARN de Transferencia/genética
18.
PLoS Comput Biol ; 15(9): e1007330, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31513579

RESUMEN

Circadian rhythms are generated by interlocked transcriptional-translational negative feedback loops (TTFLs), the molecular process implemented within a cell. The contributions, weighting and balancing between the multiple feedback loops remain debated. Dissociated, free-running dynamics in the expression of distinct clock genes has been described in recent experimental studies that applied various perturbations such as slice preparations, light pulses, jet-lag, and culture medium exchange. In this paper, we provide evidence that this "presumably transient" dissociation of circadian gene expression oscillations may occur at the single-cell level. Conceptual and detailed mechanistic mathematical modeling suggests that such dissociation is due to a weak interaction between multiple feedback loops present within a single cell. The dissociable loops provide insights into underlying mechanisms and general design principles of the molecular circadian clock.


Asunto(s)
Relojes Circadianos/genética , Ritmo Circadiano/genética , Animales , Biología Computacional , Retroalimentación , Regulación de la Expresión Génica/genética , Humanos , Ratones , Modelos Genéticos , Análisis de la Célula Individual , Neuronas del Núcleo Supraquiasmático/citología
19.
Nat Neurosci ; 22(10): 1696-1708, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31551601

RESUMEN

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process.


Asunto(s)
Envejecimiento/genética , Encéfalo/crecimiento & desarrollo , Neuronas/fisiología , Análisis de la Célula Individual , Transcriptoma/genética , Animales , Encéfalo/citología , Comunicación Celular/genética , Linaje de la Célula/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Ratones Endogámicos C57BL , Ribosomas/genética
20.
Nucleic Acids Res ; 47(19): 10452-10463, 2019 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-31552424

RESUMEN

Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.


Asunto(s)
Regulación Alostérica/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/biosíntesis , Transcripción Genética , Escherichia coli/genética , Regulación de la Expresión Génica/genética , Ligandos , Ingeniería Metabólica , Biología Sintética , Factores de Transcripción/genética
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