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1.
Mol Cell ; 78(3): 554-565.e7, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213324

RESUMO

Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of ∼20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization.


Assuntos
Cromossomos Humanos/ultraestrutura , Animais , Fator de Ligação a CCCTC/metabolismo , Células Cultivadas , Cromatina/química , Cromossomos de Mamíferos/ultraestrutura , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Humanos , Masculino , Mamíferos/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Razão Sinal-Ruído
2.
Immunity ; 48(6): 1258-1270.e6, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29884461

RESUMO

Thymus development is critical to the adaptive immune system, yet a comprehensive transcriptional framework capturing thymus organogenesis at single-cell resolution is still needed. We applied single-cell RNA sequencing (RNA-seq) to capture 8 days of thymus development, perturbations of T cell receptor rearrangement, and in vitro organ cultures, producing profiles of 24,279 cells. We resolved transcriptional heterogeneity of developing lymphocytes, and genetic perturbation confirmed T cell identity of conventional and non-conventional lymphocytes. We characterized maturation dynamics of thymic epithelial cells in vivo, classified cell maturation state in a thymic organ culture, and revealed the intrinsic capacity of thymic epithelium to preserve transcriptional regularity despite exposure to exogenous retinoic acid. Finally, by integrating the cell atlas with human genome-wide association study (GWAS) data and autoimmune-disease-related genes, we implicated embryonic thymus-resident cells as possible participants in autoimmune disease etiologies. This resource provides a single-cell transcriptional framework for biological discovery and molecular analysis of thymus organogenesis.


Assuntos
Diferenciação Celular/imunologia , Análise de Sequência de RNA/métodos , Linfócitos T/imunologia , Timo/embriologia , Animais , Doenças Autoimunes/imunologia , Embrião de Mamíferos , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Organogênese/imunologia , Linfócitos T/citologia , Timo/citologia
3.
Proc Natl Acad Sci U S A ; 120(47): e2308355120, 2023 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-37963251

RESUMO

A detailed understanding of the innate immune mechanisms involved in restricting SARS-CoV-2 infection and how the virus disrupts these processes could reveal new strategies to boost antiviral mechanisms and develop therapeutics for COVID-19. Here, we identify cellular nucleic acid-binding protein (CNBP) as a key host factor controlling SARS-CoV-2 infection. In response to RNA-sensing pathways, CNBP is phosphorylated and translocates from the cytosol to the nucleus where it binds to the interferon-ß enhancer to initiate transcription. Because SARS-CoV-2 evades immune detection by the host's RNA-sensing pathways, CNBP is largely retained in the cytosol where it restricts SARS-CoV-2 directly, leading to a battle between the host and SARS-CoV-2 that extends beyond antiviral immune signaling pathways. We further demonstrated that CNBP binds SARS-CoV-2 viral RNA directly and competes with the viral nucleocapsid protein to prevent viral RNA and nucleocapsid protein from forming liquid-liquid phase separation (LLPS) condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads, and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell-intrinsic restriction factor that disrupts LLPS to limit viral replication and spread. In addition, our studies also highlight viral condensates as important targets and strategies for the development of drugs to combat COVID-19.


Assuntos
COVID-19 , Interferons , Animais , Camundongos , Proteínas do Nucleocapsídeo , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/fisiologia , Fatores de Transcrição , Replicação Viral
4.
Proc Natl Acad Sci U S A ; 120(11): e2219523120, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36893269

RESUMO

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.


Assuntos
COVID-19 , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , COVID-19/terapia , SARS-CoV-2/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Oligonucleotídeos , Pulmão
5.
Cell ; 140(5): 704-16, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20211139

RESUMO

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.


Assuntos
Síndrome de Angelman/fisiopatologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Cognição , Humanos , Camundongos , Camundongos Knockout , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Ubiquitinação
6.
Nat Methods ; 18(9): 1046-1055, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34480151

RESUMO

Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.


Assuntos
Cromatina/química , Cromossomos Humanos/química , Reagentes de Ligações Cruzadas/química , Técnicas Genéticas , Linhagem Celular , Cromatina/metabolismo , Bases de Dados Factuais , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos
7.
Nucleic Acids Res ; 50(15): 8418-8430, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35920332

RESUMO

The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.


Assuntos
Células Endoteliais , Fibroblastos/efeitos dos fármacos , Pulmão/citologia , Oligonucleotídeos Antissenso/administração & dosagem , Animais , Fibroblastos/metabolismo , Inativação Gênica , Pulmão/efeitos dos fármacos , Camundongos , Oligonucleotídeos/administração & dosagem , Traqueia/metabolismo
9.
Development ; 146(18)2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444215

RESUMO

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.


Assuntos
Artérias/embriologia , Região Branquial/irrigação sanguínea , Sistema Cardiovascular/embriologia , Endoderma/embriologia , Morfogênese , Fator de Transcrição PAX9/metabolismo , Faringe/embriologia , Proteínas com Domínio T/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Diferenciação Celular/genética , Embrião de Mamíferos/anormalidades , Deleção de Genes , Redes Reguladoras de Genes , Heterozigoto , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Crista Neural/patologia , Fator de Transcrição PAX9/deficiência , Ligação Proteica , Transdução de Sinais
10.
Proc Natl Acad Sci U S A ; 116(36): 17970-17979, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31420514

RESUMO

Single-cell sequencing technologies have revealed an unexpectedly broad repertoire of cells required to mediate complex functions in multicellular organisms. Despite the multiple roles of adipose tissue in maintaining systemic metabolic homeostasis, adipocytes are thought to be largely homogenous with only 2 major subtypes recognized in humans so far. Here we report the existence and characteristics of 4 distinct human adipocyte subtypes, and of their respective mesenchymal progenitors. The phenotypes of these distinct adipocyte subtypes are differentially associated with key adipose tissue functions, including thermogenesis, lipid storage, and adipokine secretion. The transcriptomic signature of "brite/beige" thermogenic adipocytes reveals mechanisms for iron accumulation and protection from oxidative stress, necessary for mitochondrial biogenesis and respiration upon activation. Importantly, this signature is enriched in human supraclavicular adipose tissue, confirming that these cells comprise thermogenic depots in vivo, and explain previous findings of a rate-limiting role of iron in adipose tissue browning. The mesenchymal progenitors that give rise to beige/brite adipocytes express a unique set of cytokines and transcriptional regulators involved in immune cell modulation of adipose tissue browning. Unexpectedly, we also find adipocyte subtypes specialized for high-level expression of the adipokines adiponectin or leptin, associated with distinct transcription factors previously implicated in adipocyte differentiation. The finding of a broad adipocyte repertoire derived from a distinct set of mesenchymal progenitors, and of the transcriptional regulators that can control their development, provides a framework for understanding human adipose tissue function and role in metabolic disease.


Assuntos
Adipócitos Bege/metabolismo , Adiponectina/biossíntese , Leptina/sangue , Células-Tronco Mesenquimais/metabolismo , Termogênese , Transcriptoma , Adipócitos Bege/citologia , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/citologia
11.
Dev Biol ; 459(2): 161-180, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31862379

RESUMO

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Expressão Gênica , Genoma , Proteínas de Homeodomínio/metabolismo , Ativação Transcricional , Peixe-Zebra/embriologia , Zigoto/metabolismo , Animais , Cílios/genética , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Masculino , Proteínas de Peixe-Zebra
12.
Nat Methods ; 12(5): 401-403, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25775043

RESUMO

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.


Assuntos
Caspase 9/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Histona Desmetilases/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caspase 9/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação Enzimológica da Expressão Gênica , Genes Reporter , Histona Desmetilases/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Neisseria meningitidis/enzimologia , Proteínas Recombinantes/metabolismo
13.
Genes Dev ; 24(3): 312-26, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20123909

RESUMO

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.


Assuntos
Diferenciação Celular , Linhagem da Célula/genética , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/metabolismo , Fatores de Transcrição SOXF/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Redes Reguladoras de Genes , Proteínas HMGB/genética , Camundongos , Fatores de Transcrição SOXF/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Development ; 141(1): 219-23, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24346702

RESUMO

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Endonucleases/genética , Redes Reguladoras de Genes/genética , Células-Tronco Pluripotentes/metabolismo , Ativação Transcricional/genética , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXF/biossíntese , Transcrição Gênica
15.
Methods ; 101: 36-42, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26525193

RESUMO

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs.


Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Técnicas de Cultura de Células , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genética
16.
Stem Cells ; 31(12): 2833-41, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23765749

RESUMO

Human embryonic stem cells (hESCs) are maintained in a self-renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes using a gain-of-function screen of a large collection of human open reading frames. We identified Vestigial-like 4 (VGLL4), a cotranscriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain-of-function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. Stem Cells 2013;31:2833-2841.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/fisiologia , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Quinases Associadas a rho/metabolismo
17.
Nature ; 452(7185): 370-4, 2008 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-18354483

RESUMO

The RE1-silencing transcription factor (REST, also known as NRSF) is a master repressor of neuronal gene expression and neuronal programmes in non-neuronal lineages. Recently, REST was identified as a human tumour suppressor in epithelial tissues, suggesting that its regulation may have important physiological and pathological consequences. However, the pathways controlling REST have yet to be elucidated. Here we show that REST is regulated by ubiquitin-mediated proteolysis, and use an RNA interference (RNAi) screen to identify a Skp1-Cul1-F-box protein complex containing the F-box protein beta-TRCP (SCF(beta-TRCP)) as an E3 ubiquitin ligase responsible for REST degradation. beta-TRCP binds and ubiquitinates REST and controls its stability through a conserved phospho-degron. During neural differentiation, REST is degraded in a beta-TRCP-dependent manner. beta-TRCP is required for proper neural differentiation only in the presence of REST, indicating that beta-TRCP facilitates this process through degradation of REST. Conversely, failure to degrade REST attenuates differentiation. Furthermore, we find that beta-TRCP overexpression, which is common in human epithelial cancers, causes oncogenic transformation of human mammary epithelial cells and that this pathogenic function requires REST degradation. Thus, REST is a key target in beta-TRCP-driven transformation and the beta-TRCP-REST axis is a new regulatory pathway controlling neurogenesis.


Assuntos
Diferenciação Celular , Transformação Celular Neoplásica , Neurônios/citologia , Neurônios/patologia , Proteínas Repressoras/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , Proteínas Repressoras/genética , Especificidade por Substrato , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética
18.
Elife ; 122023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-37021774

RESUMO

Tissue-resident stem and progenitor cells are present in many adult organs, where they are important for organ homeostasis and repair in response to injury. However, the signals that activate these cells and the mechanisms governing how these cells renew or differentiate are highly context-dependent and incompletely understood, particularly in non-hematopoietic tissues. In the skin, melanocyte stem and progenitor cells are responsible for replenishing mature pigmented melanocytes. In mammals, these cells reside in the hair follicle bulge and bulb niches where they are activated during homeostatic hair follicle turnover and following melanocyte destruction, as occurs in vitiligo and other skin hypopigmentation disorders. Recently, we identified melanocyte progenitors in adult zebrafish skin. To elucidate mechanisms governing melanocyte progenitor renewal and differentiation we analyzed individual transcriptomes from thousands of melanocyte lineage cells during the regeneration process. We identified transcriptional signatures for progenitors, deciphered transcriptional changes and intermediate cell states during regeneration, and analyzed cell-cell signaling changes to discover mechanisms governing melanocyte regeneration. We identified KIT signaling via the RAS/MAPK pathway as a regulator of melanocyte progenitor direct differentiation and asymmetric division. Our findings show how activation of different subpopulations of mitfa-positive cells underlies cellular transitions required to properly reconstitute the melanocyte pigmentary system following injury.


Assuntos
Melanócitos , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Melanócitos/metabolismo , Pele , Células-Tronco/metabolismo , Folículo Piloso , Transdução de Sinais , Diferenciação Celular , Mamíferos
19.
Dev Cell ; 58(18): 1801-1818.e15, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37751684

RESUMO

Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.


Assuntos
Faringe , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Endoderma/metabolismo , Sistema Digestório , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento
20.
Elife ; 122023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36719724

RESUMO

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation.


Assuntos
RNA Longo não Codificante , Gravidez , Feminino , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transição Epitelial-Mesenquimal , Endoderma , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Diferenciação Celular/genética
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