RESUMO
The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury-repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the 'pulmonary ionocyte', which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.
Assuntos
Brônquios/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Análise de Célula Única , Traqueia/citologia , Adolescente , Adulto , Animais , Diferenciação Celular/genética , Células Cultivadas , Criança , Pré-Escolar , Fibrose Cística/genética , Feminino , Imunofluorescência , Fatores de Transcrição Forkhead/metabolismo , Homeostase/genética , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Receptores Notch/metabolismo , Regeneração/genética , Análise de Sequência de RNA , Transdução de Sinais/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Adulto JovemRESUMO
A limited understanding of the pathology underlying chronic wounds has hindered the development of effective diagnostic markers and pharmaceutical interventions. This study aimed to elucidate the molecular composition of various common chronic ulcer types to facilitate drug discovery strategies. We conducted a comprehensive analysis of leg ulcers (LUs), encompassing venous and arterial ulcers, foot ulcers (FUs), pressure ulcers (PUs), and compared them with surgical wound healing complications (WHCs). To explore the pathophysiological mechanisms and identify similarities or differences within wounds, we dissected wounds into distinct subregions, including the wound bed, border, and peri-wound areas, and compared them against intact skin. By correlating histopathology, RNA sequencing (RNA-Seq), and immunohistochemistry (IHC), we identified unique genes, pathways, and cell type abundance patterns in each wound type and subregion. These correlations aim to aid clinicians in selecting targeted treatment options and informing the design of future preclinical and clinical studies in wound healing. Notably, specific genes, such as PITX1 and UPP1, exhibited exclusive upregulation in LUs and FUs, potentially offering significant benefits to specialists in limb preservation and clinical treatment decisions. In contrast, comparisons between different wound subregions, regardless of wound type, revealed distinct expression profiles. The pleiotropic chemokine-like ligand GPR15L (C10orf99) and transmembrane serine proteases TMPRSS11A/D were significantly upregulated in wound border subregions. Interestingly, WHCs exhibited a nearly identical transcriptome to PUs, indicating clinical relevance. Histological examination revealed blood vessel occlusions with impaired angiogenesis in chronic wounds, alongside elevated expression of genes and immunoreactive markers related to blood vessel and lymphatic epithelial cells in wound bed subregions. Additionally, inflammatory and epithelial markers indicated heightened inflammatory responses in wound bed and border subregions and reduced wound bed epithelialization. In summary, chronic wounds from diverse anatomical sites share common aspects of wound pathophysiology but also exhibit distinct molecular differences. These unique molecular characteristics present promising opportunities for drug discovery and treatment, particularly for patients suffering from chronic wounds. The identified diagnostic markers hold the potential to enhance preclinical and clinical trials in the field of wound healing.
Assuntos
Pé Diabético , Úlcera da Perna , Úlcera por Pressão , Lesões dos Tecidos Moles , Humanos , Úlcera por Pressão/genética , Úlcera por Pressão/terapia , Pé Diabético/terapia , Úlcera da Perna/terapia , Expressão Gênica , SupuraçãoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores de RNA/metabolismo , Sarcoma de Ewing/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fenótipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Piperazinas/farmacologia , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Sarcoma de Ewing/tratamento farmacológicoRESUMO
PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.
Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Genoma Humano/genética , Mitofagia/genética , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Células Cultivadas , Células HCT116 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Neurônios/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: RNA-sequencing (RNA-seq) has emerged as one of the most sensitive tool for gene expression analysis. Among the library preparation methods available, the standard poly(A) + enrichment provides a comprehensive, detailed, and accurate view of polyadenylated RNAs. However, on samples of suboptimal quality ribosomal RNA depletion and exon capture methods have recently been reported as better alternatives. METHODS: We compared for the first time three commercial Illumina library preparation kits (TruSeq Stranded mRNA, TruSeq Ribo-Zero rRNA Removal, and TruSeq RNA Access) as representatives of these three different approaches using well-established human reference RNA samples from the MAQC/SEQC consortium on a wide range of input amounts (from 100 ng down to 1 ng) and degradation levels (intact, degraded, and highly degraded). RESULTS: We assessed the accuracy of the generated expression values by comparison to gold standard TaqMan qPCR measurements and gained unprecedented insight into the limits of applicability in terms of input quantity and sample quality of each protocol. We found that each protocol generates highly reproducible results (R 2 > 0.92) on intact RNA samples down to input amounts of 10 ng. For degraded RNA samples, Ribo-Zero showed clear performance advantages over the other two protocols as it generated more accurate and better reproducible gene expression results even at very low input amounts such as 1 ng and 2 ng. For highly degraded RNA samples, RNA Access performed best generating reliable data down to 5 ng input. CONCLUSIONS: We found that the ribosomal RNA depletion protocol from Illumina works very well at amounts far below recommendation and over a good range of intact and degraded material. We also infer that the exome-capture protocol (RNA Access, Illumina) performs better than other methods on highly degraded and low amount samples.
Assuntos
Análise de Sequência de RNA/métodos , Humanos , Controle de Qualidade , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Taq Polimerase/metabolismoRESUMO
BACKGROUND: Hearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss. RESULTS: In order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach. CONCLUSION: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.
Assuntos
Transdiferenciação Celular , Células Ciliadas Auditivas Internas/citologia , MicroRNAs/metabolismo , Órgão Espiral/citologia , Regeneração , Animais , Linhagem Celular , Técnicas de Introdução de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/genética , Análise de Sequência de RNARESUMO
BACKGROUND: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge. RESULTS: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation. CONCLUSIONS: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.
Assuntos
Sistemas CRISPR-Cas , Transformação Celular Neoplásica/genética , Estudo de Associação Genômica Ampla , Mutação , Oncogenes , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Atopic dermatitis (AD) is a debilitating inflammatory skin disorder. Biologics targeting the IL-4/IL-13 axis are effective in AD, but there is still a large proportion of patients who do not respond to IL-4R blockade. Further exploration of potentially pathogenic T-cell-derived cytokines in AD may lead to new effective treatments. This study aimed to investigate the downstream effects of IL-26 on skin in the context of type 2 skin inflammation. We found that IL-26 alone exhibited limited inflammatory activity in the skin. However, in the presence of IL-1ß, IL-26 potentiated the secretion of TSLP, CXCL1, and CCL20 from human epidermis through Jak/signal transducer and activator of transcription signaling. Moreover, in an in vivo AD-like skin inflammation model, IL-26 exacerbated skin pathology and locally increased type 2 cytokines, most notably of IL13 in skin T helper cells. Neutralization of IL-1ß abrogated IL-26-mediated effects, indicating that the presence of IL-1ß is required for full IL-26 downstream action in vivo. These findings suggest that the presence of IL-1ß enables IL-26 to be a key amplifier of inflammation in the skin. As such, IL-26 may contribute to the development and pathogenesis of inflammatory skin disorders such as AD.
Assuntos
Dermatite Atópica , Interleucina-1beta , Interleucinas , Humanos , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Interleucina-1beta/metabolismo , Animais , Camundongos , Interleucinas/metabolismo , Interleucinas/imunologia , Modelos Animais de Doenças , Citocinas/metabolismo , Transdução de Sinais/imunologia , Feminino , Queratinócitos/imunologia , Queratinócitos/metabolismo , Pele/patologia , Pele/imunologia , Células CultivadasRESUMO
ARID1B is a SWI/SNF subunit frequently mutated in human Coffin-Siris syndrome (CSS) and it is necessary for proliferation of ARID1A mutant cancers. While most CSS ARID1B aberrations introduce frameshifts or stop codons, the functional consequence of missense mutations found in ARID1B is unclear. We here perform saturated mutagenesis screens on ARID1B and demonstrate that protein destabilization is the main mechanism associated with pathogenic missense mutations in patients with Coffin-Siris Syndrome.
Assuntos
Proteínas de Ligação a DNA , Deformidades Congênitas da Mão , Deficiência Intelectual , Micrognatismo , Mutação de Sentido Incorreto , Estabilidade Proteica , Fatores de Transcrição , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Micrognatismo/genética , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Anormalidades Múltiplas/genética , Face/anormalidades , Pescoço/anormalidadesRESUMO
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world's population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.
RESUMO
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Assuntos
Anemia Falciforme , Antidrepanocíticos , Hemoglobina Fetal , Fatores de Transcrição Kruppel-Like , Proteínas do Tecido Nervoso , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Cristalografia por Raios X , Descoberta de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Macaca fascicularis , Proteínas do Tecido Nervoso/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
YAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neoplasias , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Sinalização YAP , Epigenômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução de Sinais/genéticaRESUMO
Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the huntingtin (HTT) gene. The mutant HTT (mHTT) protein causes neuronal dysfunction, causing progressive motor, cognitive and behavioral abnormalities. Current treatments for HD only alleviate symptoms, but cerebral spinal fluid (CSF) or central nervous system (CNS) delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA-induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. Here, we present an alternative disease modifying therapy the orally available, brain penetrant small molecule branaplam. By promoting inclusion of a pseudoexon in the primary transcript, branaplam lowers mHTT protein levels in HD patient cells, in an HD mouse model and in blood samples from Spinal Muscular Atrophy (SMA) Type I patients dosed orally for SMA (NCT02268552). Our work paves the way for evaluating branaplam's utility as an HD therapy, leveraging small molecule splicing modulators to reduce expression of dominant disease genes by driving pseudoexon inclusion.
Assuntos
Doença de Huntington , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Doença de Huntington/metabolismo , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
Fibrosis is characterized by the excessive production of collagen and other extracellular matrix (ECM) components and represents a leading cause of morbidity and mortality worldwide. Previous studies of nonalcoholic steatohepatitis (NASH) with fibrosis were largely restricted to bulk transcriptome profiles. Thus, our understanding of this disease is limited by an incomplete characterization of liver cell types in general and hepatic stellate cells (HSCs) in particular, given that activated HSCs are the major hepatic fibrogenic cell population. To help fill this gap, we profiled 17,810 non-parenchymal cells derived from six healthy human livers. In conjunction with public single-cell data of fibrotic/cirrhotic human livers, these profiles enable the identification of potential intercellular signaling axes (e.g., ITGAV-LAMC1, TNFRSF11B-VWF and NOTCH2-DLL4) and master regulators (e.g., RUNX1 and CREB3L1) responsible for the activation of HSCs during fibrogenesis. Bulk RNA-seq data of NASH patient livers and rodent models for liver fibrosis of diverse etiologies allowed us to evaluate the translatability of candidate therapeutic targets for NASH-related fibrosis. We identified 61 liver fibrosis-associated genes (e.g., AEBP1, PRRX1 and LARP6) that may serve as a repertoire of translatable drug target candidates. Consistent with the above regulon results, gene regulatory network analysis allowed the identification of CREB3L1 as a master regulator of many of the 61 genes. Together, this study highlights potential cell-cell interactions and master regulators that underlie HSC activation and reveals genes that may represent prospective hallmark signatures for liver fibrosis.
Assuntos
Células Estreladas do Fígado , Hepatopatia Gordurosa não Alcoólica , Transcriptoma , Animais , Voluntários Saudáveis , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Análise de Célula ÚnicaRESUMO
Inflammatory responses are crucial for regeneration following peripheral nerve injury (PNI). PNI triggers inflammatory responses at the site of injury. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) sense foreign and self-DNA and trigger type I interferon (IFN) immune responses. We demonstrate here that following PNI, the cGAS/STING pathway is upregulated in the sciatic nerve of naive rats and dysregulated in old rats. In a nerve crush mouse model where STING is knocked out, myelin content in sciatic nerve is increased resulting in accelerated functional axon recovery. STING KO mice have lower macrophage number in sciatic nerve and decreased microglia activation in spinal cord 1 week post injury. STING activation regulated processing of colony stimulating factor 1 receptor (CSF1R) and microglia survival in vitro. Taking together, these data highlight a previously unrecognized role of STING in the regulation of nerve regeneration.
RESUMO
BACKGROUND & AIMS: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics. METHODS: Chronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen. RESULTS: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis. CONCLUSIONS: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.
Assuntos
Tetracloreto de Carbono/toxicidade , Redes Reguladoras de Genes/efeitos dos fármacos , Predisposição Genética para Doença , Cirrose Hepática/induzido quimicamente , Fígado/patologia , Animais , Tetracloreto de Carbono/administração & dosagem , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Humanos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Locos de Características QuantitativasRESUMO
Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Redes Reguladoras de Genes , Carcinogênese/genética , Epigenômica , Genoma Humano , Humanos , Elementos Reguladores de TranscriçãoRESUMO
Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , DNA/genética , Células A549 , Animais , Linhagem Celular Tumoral , Eletroporação , Fibroblastos/metabolismo , Genoma Humano , Células HCT116 , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , RNA Guia de Cinetoplastídeos/metabolismo , Ratos , Pele/metabolismoRESUMO
A promising approach for the treatment of nonalcoholic steatohepatitis (NASH) is the inhibition of enhanced hepatic de novo lipogenesis (DNL), which is the synthesis of fatty acids from nonlipid sources. This study assesses three approaches to DNL suppression in a newly developed dietary NASH mouse model: i) dietary intervention (switch from NASH-inducing diet to normal diet); ii) inhibition of acetyl-coenzyme A carboxylase (ACC), the enzyme catalyzing the rate-limiting step in DNL; and iii) activation of farnesoid X receptor (FXR), a major transcriptional regulator of DNL. C57BL/6J mice on a high-fat diet combined with ad libitum consumption of a fructose-sucrose solution developed several of the liver histologic features seen in human disease, including steatosis, inflammation, and fibrosis, accompanied by elevated fibrosis biomarkers and liver injury enzymes. Obesity and metabolic impairments were associated with increased intestinal permeability and progression to adenoma and hepatocellular carcinoma. All three approaches led to resolution of established NASH with fibrosis in mice; however, some differences were noted, e.g., with respect to the degree of hepatic steatosis attenuation. While ACC inhibition resulted in elevated blood triglycerides and peripheral obesity, FXR activation prevented peripheral obesity in NASH mice. Comparative transcriptome analysis underlined the translatability of the mouse model to human NASH and revealed novel mechanistic insights into differential regulation of lipid, inflammatory, and extracellular matrix pathways by FXR agonism and ACC inhibition. Conclusion: Novel insights are provided on back translation of clinically observed endpoints of DNL inhibition by targeting ACC or FXR, which are promising therapeutic options for the treatment of NASH, in a newly developed diet-induced NASH mouse model.