RESUMO
Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.
Assuntos
Cromatina , Redes Reguladoras de Genes , Animais , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
T lymphocytes, the major effector cells in cellular immunity, produce cytokines in immune responses to mediate inflammation and regulate other types of immune cells. Work in the last three decades has revealed significant heterogeneity in CD4+ T cells, in terms of their cytokine expression, leading to the discoveries of T helper 1 (Th1), Th2, Th17, and T follicular helper (Tfh) cell subsets. These cells possess unique developmental and regulatory pathways and play distinct roles in immunity and immune-mediated pathologies. Other types of T cells, including regulatory T cells and γδ T cells, as well as innate lymphocytes, display similar features of subpopulations, which may play differential roles in immunity. Mechanisms exist to prevent cytokine production by T cells to maintain immune tolerance to self-antigens, some of which may also underscore immune exhaustion in the context of tumors. Understanding cytokine regulation and function has offered innovative treatment of many human diseases.
Assuntos
Citocinas , Linfócitos T Reguladores , Animais , Humanos , Tolerância Imunológica , Imunidade Celular , Linfócitos T Auxiliares-Indutores , Células Th17RESUMO
A fundamental question in developmental immunology is how bipotential thymocyte precursors generate both CD4+ helper and CD8+ cytotoxic T cell lineages. The MHC specificity of αß T cell receptors (TCRs) on precursors is closely correlated with cell fate-determining processes, prompting studies to characterize how variations in TCR signaling are linked with genetic programs establishing lineage-specific gene expression signatures, such as exclusive CD4 or CD8 expression. The key transcription factors ThPOK and Runx3 have been identified as mediating development of helper and cytotoxic T cell lineages, respectively. Together with increasing knowledge of epigenetic regulators, these findings have advanced our understanding of the transcription factor network regulating the CD4/CD8 dichotomy. It has also become apparent that CD4+ T cells retain developmental plasticity, allowing them to acquire cytotoxic activity in the periphery. Despite such advances, further studies are necessary to identify the molecular links between TCR signaling and the nuclear machinery regulating expression of ThPOK and Runx3.
Assuntos
Diferenciação Celular/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos CD4/genética , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Imunomodulação/genética , Imunomodulação/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Sequências Reguladoras de Ácido Nucleico , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Assuntos
Cisteína , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cisteína/metabolismo , Cisteína/química , Ligantes , Melanoma/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Oxirredução , Transdução de Sinais , Fatores de Transcrição SOXE/química , Fatores de Transcrição SOXE/metabolismoRESUMO
The dendritic cells (DCs) of the immune system function in innate and adaptive responses by directing activity of various effector cells rather than serving as effectors themselves. DCs and closely related myeloid lineages share expression of many surface receptors, presenting a challenge in distinguishing their unique in vivo functions. Recent work has taken advantage of unique transcriptional programs to identify and manipulate murine DCs in vivo. This work has assigned several nonredundant in vivo functions to distinct DC lineages, consisting of plasmacytoid DCs and several subsets of classical DCs that promote different immune effector modules in response to pathogens. In parallel, a correspondence between human and murine DC subsets has emerged, underlying structural similarities for the DC lineages between these species. Recent work has begun to unravel the transcriptional circuitry that controls the development and diversification of DCs from common progenitors in the bone marrow.
Assuntos
Células da Medula Óssea/fisiologia , Células Dendríticas/fisiologia , Regulação da Expressão Gênica , Imunidade Celular , Animais , Diferenciação Celular , Linhagem da Célula , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Imunidade Celular/genética , Camundongos , Ativação TranscricionalRESUMO
The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.
Assuntos
Diferenciação Celular , Hipersensibilidade/imunologia , Imunidade Inata , Linfócitos/imunologia , Células Progenitoras Linfoides/imunologia , Animais , Linhagem da Célula , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Altered microglial states affect neuroinflammation, neurodegeneration, and disease but remain poorly understood. Here, we report 194,000 single-nucleus microglial transcriptomes and epigenomes across 443 human subjects and diverse Alzheimer's disease (AD) pathological phenotypes. We annotate 12 microglial transcriptional states, including AD-dysregulated homeostatic, inflammatory, and lipid-processing states. We identify 1,542 AD-differentially-expressed genes, including both microglia-state-specific and disease-stage-specific alterations. By integrating epigenomic, transcriptomic, and motif information, we infer upstream regulators of microglial cell states, gene-regulatory networks, enhancer-gene links, and transcription-factor-driven microglial state transitions. We demonstrate that ectopic expression of our predicted homeostatic-state activators induces homeostatic features in human iPSC-derived microglia-like cells, while inhibiting activators of inflammation can block inflammatory progression. Lastly, we pinpoint the expression of AD-risk genes in microglial states and differential expression of AD-risk genes and their regulators during AD progression. Overall, we provide insights underlying microglial states, including state-specific and AD-stage-specific microglial alterations at unprecedented resolution.
Assuntos
Doença de Alzheimer , Microglia , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Regulação da Expressão Gênica , Inflamação/patologia , Microglia/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , EpigenomaRESUMO
Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.
Assuntos
Montagem e Desmontagem da Cromatina , Complexos Multiproteicos , Proteínas Nucleares , Humanos , Cromatina , Proteínas de Ligação a DNA/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.
Assuntos
Cromatina , Nucleossomos , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Nucleossomos/genética , Fatores de Transcrição/genéticaRESUMO
The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function.
Assuntos
Imunidade Adaptativa/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica , Imunidade Inata/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Transcrição Gênica , Animais , Diferenciação Celular , Humanos , Linfócitos/citologia , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismoRESUMO
Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.
Assuntos
DNA/química , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Iniciação da Transcrição Genética , Sequência de Aminoácidos , Microscopia Crioeletrônica , DNA/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Polimerase II/ultraestrutura , Deleção de Sequência , Fator de Transcrição TFIIH , Fatores de Transcrição TFII/metabolismoRESUMO
Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.
Assuntos
Elementos de DNA Transponíveis/genética , Análise de Célula Única/métodos , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Imunoprecipitação da Cromatina , Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Ligação Proteica , Análise de Sequência de RNA , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.
Assuntos
Bancos de Espécimes Biológicos , Tumores Neuroendócrinos/patologia , Organoides/patologia , Animais , Cromossomos Humanos/genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Masculino , Camundongos , Modelos Genéticos , Mutação/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transcriptoma/genética , Sequenciamento Completo do GenomaRESUMO
The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.
Assuntos
Neoplasias da Mama/genética , Metástase Neoplásica/genética , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteína Homeobox Nanog/metabolismo , Metástase Neoplásica/fisiopatologia , Células Neoplásicas Circulantes/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3RESUMO
Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.
Assuntos
Evolução Molecular Direcionada/métodos , Regulação Alostérica , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Mutação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Sindbis virus/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plants constantly perceive internal and external cues, many of which they need to address to safeguard their proper development and survival. They respond to these cues by selective activation of specific metabolic pathways involving a plethora of molecular players that act and interact in complex networks. In this review, we illustrate and discuss the complexity in the combinatorial control of plant specialized metabolism. We hereby go beyond the intuitive concept of combinatorial control as exerted by modular-acting complexes of transcription factors that govern expression of specialized metabolism genes. To extend this discussion, we also consider all known hierarchical levels of regulation of plant specialized metabolism and their interfaces by referring to reported regulatory concepts from the plant field. Finally, we speculate on possible yet-to-be-discovered regulatory principles of plant specialized metabolism that are inspired by knowledge from other kingdoms of life and areas of biological research.
Assuntos
Plantas/metabolismo , Evolução Biológica , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica , Plantas/genética , Transdução de SinaisRESUMO
Non-coding regions comprise most of the human genome and harbor a significant fraction of risk alleles for neuropsychiatric diseases, yet their functions remain poorly defined. We created a high-resolution map of non-coding elements involved in human cortical neurogenesis by contrasting chromatin accessibility and gene expression in the germinal zone and cortical plate of the developing cerebral cortex. We link distal regulatory elements (DREs) to their cognate gene(s) together with chromatin interaction data and show that target genes of human-gained enhancers (HGEs) regulate cortical neurogenesis and are enriched in outer radial glia, a cell type linked to human cortical evolution. We experimentally validate the regulatory effects of predicted enhancers for FGFR2 and EOMES. We observe that common genetic variants associated with educational attainment, risk for neuropsychiatric disease, and intracranial volume are enriched within regulatory elements involved in cortical neurogenesis, demonstrating the importance of this early developmental process for adult human cognitive function.
Assuntos
Córtex Cerebral/metabolismo , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese , Neurônios/metabolismo , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Feminino , Humanos , Masculino , Neurônios/citologia , Polimorfismo Genético , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , RNA , Anêmonas-do-Mar/fisiologia , Actinas/química , Motivos de Aminoácidos , Animais , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Genoma , Genômica , Filogenia , Anêmonas-do-Mar/genética , Análise de Sequência de RNA , Transcriptoma , Tubulina (Proteína)/químicaRESUMO
Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Edição de Genes/métodos , Fatores de Transcrição/genética , Animais , Núcleo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Fatores de Transcrição/metabolismoRESUMO
Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.