RESUMO
Nuclear hormone receptors (NHRs) are a family of ligand-regulated transcription factors that control key aspects of development and physiology. The regulation of NHRs by ligands derived from metabolism or diet makes them excellent pharmacological targets, and the mechanistic understanding of how NHRs interact with their ligands to regulate downstream gene networks, along with the identification of ligands for orphan NHRs, could enable innovative approaches for cellular engineering, disease modeling and regenerative medicine. We review recent discoveries in the identification of physiologic ligands for NHRs. We propose new models of ligand-receptor co-evolution, the emergence of hormonal function and models of regulation of NHR specificity and activity via one-ligand and two-ligand models as well as feedback loops. Lastly, we discuss limitations on the processes for the identification of physiologic NHR ligands and emerging new methodologies that could be used to identify the natural ligands for the remaining 17 orphan NHRs in the human genome.
Assuntos
Metaboloma , Receptores Citoplasmáticos e Nucleares , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Ligantes , AnimaisRESUMO
Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren's syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.
Assuntos
Diferenciação Celular , Disautonomia Familiar , Células-Tronco Pluripotentes , Humanos , Células-Tronco Pluripotentes/citologia , Disautonomia Familiar/patologia , Neurônios , Síndrome de Sjogren/patologia , COVID-19/virologia , COVID-19/patologia , Animais , Sistema Nervoso Parassimpático , Células de Schwann , Camundongos , SARS-CoV-2/fisiologiaRESUMO
Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Organogênese/efeitos dos fármacos , Esfingolipídeos/farmacologia , Animais , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Vasos Linfáticos/efeitos dos fármacos , Camundongos , Organogênese/fisiologia , Proteínas Repressoras/fisiologiaRESUMO
In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.
Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Proliferação de Células , Córtex Cerebral/embriologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Divisão Celular/genética , Movimento Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Regulação para Baixo/genética , Proteínas da Matriz Extracelular/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Microscopia de Vídeo , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Neurônios/citologia , Interferência de RNA/fisiologia , Proteína Reelina , Serina Endopeptidases/genética , Células-Tronco/citologia , Fatores de TempoRESUMO
Nuclear hormone receptors are a family of transcription factors regulated by small molecules derived from the endogenous metabolism or diet. There are forty-eight nuclear hormone receptors in the human genome, twenty of which are still orphans. In this review, we make a brief historical journey from the first observations by Berthold in 1849 to the era of orphan receptors that began with the sequencing of the Caenorhabditis elegans genome in 1998. We discuss the evolution of nuclear hormone receptors and the putative ancestral ligands as well as how the ligand universe has expanded over time. This leads us to define four classes of metabolites-fatty acids, terpenoids, porphyrins and amino acid derivatives-that generate all known ligands for nuclear hormone receptors. We conclude by discussing the ongoing efforts to identify new classes of ligands for orphan receptors.
Assuntos
Ligantes , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Animais , Biologia/história , Caenorhabditis elegans , Ácidos Graxos/metabolismo , Genoma Humano , História do Século XIX , História do Século XX , História do Século XXI , Hormônios/metabolismo , Humanos , Modelos Biológicos , Terpenos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes/métodos , Células-Tronco Embrionárias Humanas/fisiologia , Células Cultivadas , Doxiciclina/farmacologia , Regulação da Expressão Gênica , Teste de Complementação Genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Fatores de Transcrição SOXF/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , TransgenesRESUMO
The high-dimensional data created by high-throughput technologies require visualization tools that reveal data structure and patterns in an intuitive form. We present PHATE, a visualization method that captures both local and global nonlinear structure using an information-geometric distance between data points. We compare PHATE to other tools on a variety of artificial and biological datasets, and find that it consistently preserves a range of patterns in data, including continual progressions, branches and clusters, better than other tools. We define a manifold preservation metric, which we call denoised embedding manifold preservation (DEMaP), and show that PHATE produces lower-dimensional embeddings that are quantitatively better denoised as compared to existing visualization methods. An analysis of a newly generated single-cell RNA sequencing dataset on human germ-layer differentiation demonstrates how PHATE reveals unique biological insight into the main developmental branches, including identification of three previously undescribed subpopulations. We also show that PHATE is applicable to a wide variety of data types, including mass cytometry, single-cell RNA sequencing, Hi-C and gut microbiome data.
Assuntos
Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Animais , Big Data , Diferenciação Celular , Células Cultivadas , Simulação por Computador , Bases de Dados Genéticas , Microbioma Gastrointestinal , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
As somatic cells are converted into induced pluripotent stem cells (iPSCs), their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratios, DNA methylation patterns, and enhancer and promoter status. The molecular machinery underlying this process is largely unknown. Here, we show that embryonic stem cell (ESC)-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer, and are required for efficient reprogramming of mouse and human cells. When co-expressed with Oct4, Klf4, Sox2, and Myc (OKSM) factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in 2 to 4 days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway and contribute to downregulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our work provides critical insights into how the epigenome is remodeled during acquisition of pluripotency.
Assuntos
Reprogramação Celular , Epigênese Genética , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fatores de TranscriçãoRESUMO
Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, referred to as Polycomb domains. Non-canonical PRC1 complexes retain RING1/RNF2 E3-ubiquitin ligases but have unique sets of accessory subunits. How these non-canonical complexes recognize and regulate their gene targets remains poorly understood. Here, we show that the BCL6 co-repressor (BCOR), a member of the PRC1.1 complex, is critical for maintaining primed pluripotency in human embryonic stem cells (ESCs). BCOR depletion leads to the erosion of Polycomb domains at key developmental loci and the initiation of differentiation along endoderm and mesoderm lineages. The C terminus of BCOR regulates the assembly and targeting of the PRC1.1 complex, while the N terminus contributes to BCOR-PRC1.1 repressor function. Our findings advance understanding of Polycomb targeting and repression in ESCs and could apply broadly across developmental systems.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Complexos Multiproteicos/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Cromatina/metabolismo , Proteínas F-Box/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Metilação , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/químicaRESUMO
Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.
Assuntos
Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Sistema Nervoso/citologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , RNA/genética , Proteínas Repressoras/fisiologia , Proteína do Retinoblastoma/fisiologia , Células-Tronco/citologia , Animais , Proliferação de Células , Eletroporação , Feminino , Camundongos , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genéticaRESUMO
Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.