RESUMO
In this work, we describe NEAT-seq (sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells), enabling interrogation of regulatory mechanisms spanning the central dogma. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive T cell subsets and identify examples of TFs with regulatory activity gated by transcription, translation and regulation of chromatin binding. We also link a noncoding genome-wide association study single-nucleotide polymorphism (SNP) within a GATA motif to a putative target gene, using NEAT-seq data to internally validate SNP impact on GATA3 regulation.
Assuntos
Cromatina , Proteínas Nucleares , Cromatina/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Proteínas Nucleares/genética , TranscriptomaRESUMO
Inflammatory bowel disease is associated with increased reactive oxygen species (ROS) and decreased antioxidant response in the intestinal mucosa. Expression of the mitochondrial protein prohibitin (PHB) is also decreased during intestinal inflammation. Our previous study showed that genetic restoration of colonic epithelial PHB expression [villin-PHB transgenic (PHB Tg) mice] attenuated dextran sodium sulfate (DSS)-induced colitis/oxidative stress and sustained expression of colonic nuclear factor erythroid 2-related factor 2 (Nrf2), a cytoprotective transcription factor. This study investigated the role of Nrf2 in mediating PHB-induced protection against colitis and expression of the antioxidant response element (ARE)-regulated antioxidant genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1). PHB-transfected Caco-2-BBE human intestinal epithelial cells maintained increased ARE activation and decreased intracellular ROS levels compared with control vector-transfected cells during Nrf2 knockdown by small interfering RNA. Treatment with the ERK inhibitor PD-98059 decreased PHB-induced ARE activation, suggesting that ERK constitutes a significant portion of PHB-mediated ARE activation in Caco-2-BBE cells. PHB Tg, Nrf2(-/-), and PHB Tg/Nrf2(-/-) mice were treated with DSS or 2,4,6-trinitrobenzene sulfonic acid (TNBS), and inflammation and expression of HO-1 and NQO-1 were assessed. PHB Tg/Nrf2(-/-) mice mimicked PHB Tg mice, with attenuated DSS- or TNBS-induced colitis and induction of colonic HO-1 and NQO-1 expression, despite deletion of Nrf2. PHB Tg/Nrf2(-/-) mice exhibited increased activation of ERK during colitis. Our results suggest that maintaining expression of intestinal epithelial cell PHB, which is decreased during colitis, reduces the severity of inflammation and increases colonic levels of the antioxidants HO-1 and NQO-1 via a mechanism independent of Nrf2.
Assuntos
Colite/genética , Colite/prevenção & controle , Subunidade p45 do Fator de Transcrição NF-E2/fisiologia , Proteínas Repressoras/fisiologia , Animais , Elementos de Resposta Antioxidante/fisiologia , Western Blotting , Células CACO-2 , Linhagem Celular , Colite/induzido quimicamente , Sulfato de Dextrana , Genes Reporter , Heme Oxigenase-1/metabolismo , Humanos , Luciferases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/metabolismo , Subunidade p45 do Fator de Transcrição NF-E2/genética , Peroxidase/metabolismo , Proibitinas , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell typespecific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, and BCL6) and transcription factors regulating B cell differentiation (POU2AF1 and HHEX). Together, these analyses provide a powerful new cell typeresolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.
Assuntos
Autoimunidade/imunologia , Centro Germinativo/imunologia , Proteínas de Homeodomínio/imunologia , Interleucinas/imunologia , Análise de Célula Única , Transativadores/imunologia , Fatores de Transcrição/imunologia , Diferenciação Celular/imunologia , Epigenômica , Proteínas de Homeodomínio/genética , Humanos , Interleucinas/genética , Tonsila Palatina/imunologia , Análise de Sequência de RNA , Transativadores/genética , Fatores de Transcrição/genética , TranscriptomaRESUMO
Identifying the causes of human diseases requires deconvolution of abnormal molecular phenotypes spanning DNA accessibility, gene expression and protein abundance1-3. We present a single-cell framework that integrates highly multiplexed protein quantification, transcriptome profiling and analysis of chromatin accessibility. Using this approach, we establish a normal epigenetic baseline for healthy blood development, which we then use to deconvolve aberrant molecular features within blood from patients with mixed-phenotype acute leukemia4,5. Despite widespread epigenetic heterogeneity within the patient cohort, we observe common malignant signatures across patients as well as patient-specific regulatory features that are shared across phenotypic compartments of individual patients. Integrative analysis of transcriptomic and chromatin-accessibility maps identified 91,601 putative peak-to-gene linkages and transcription factors that regulate leukemia-specific genes, such as RUNX1-linked regulatory elements proximal to the marker gene CD69. These results demonstrate how integrative, multiomic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples.
Assuntos
Cromatina/genética , Leucemia Aguda Bifenotípica/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Células da Medula Óssea/citologia , Cromatina/química , Análise por Conglomerados , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Epigênese Genética/genética , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
Carcinoma Ductal Pancreático/patologia , Perfilação da Expressão Gênica/métodos , Neoplasias Pancreáticas/patologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Análise de Sequência de RNA/métodos , Regulação para Cima , Animais , Carcinoma Ductal Pancreático/genética , Hipóxia Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Engenharia Genética , Humanos , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Microambiente TumoralRESUMO
INTRODUCTION: Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells. METHODS: We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1) or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. RESULTS: TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability. CONCLUSIONS: Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.
Assuntos
Autofagia , Colo/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Acetilcisteína/farmacologia , Animais , Autofagia/efeitos dos fármacos , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Proibitinas , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.