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Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.
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Biological sex is an important risk factor in cancer, but the underlying cell types and mechanisms remain obscure. Since tumor development is regulated by the immune system, we hypothesize that sex-biased immune interactions underpin sex differences in cancer. The male-biased glioblastoma multiforme (GBM) is an aggressive and treatment-refractory tumor in urgent need of more innovative approaches, such as considering sex differences, to improve outcomes. GBM arises in the specialized brain immune environment dominated by microglia, so we explored sex differences in this immune cell type. We isolated adult human TAM-MGs (tumor-associated macrophages enriched for microglia) and control microglia and found sex-biased inflammatory signatures in GBM and lower-grade tumors associated with pro-tumorigenic activity in males and anti-tumorigenic activity in females. We demonstrated that genes expressed or modulated by the inactive X chromosome facilitate this bias. Together, our results implicate TAM-MGs, specifically their sex chromosomes, as drivers of male bias in GBM.
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Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.
Assuntos
Carcinoma de Células Acinares , Carcinoma Adenoide Cístico , Carcinoma , Neoplasias das Glândulas Salivares , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Carcinoma Adenoide Cístico/patologia , Carcinoma/patologia , Carcinoma de Células Acinares/metabolismo , RNA/metabolismoRESUMO
Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.
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Síndrome da Persistência do Padrão de Circulação Fetal , Recém-Nascido , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Redes Reguladoras de Genes/genética , Fator A de Crescimento do Endotélio Vascular/genética , Células Endoteliais/patologia , Multiômica , Pulmão/patologia , RNA , Fatores de Transcrição Forkhead/genéticaRESUMO
In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.
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Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Adulto , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Cromatina/genética , Transcriptoma , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Mutação , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismoRESUMO
Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.
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Diabetes Mellitus Tipo 1/genética , Epigenômica , Predisposição Genética para Doença , Análise de Célula Única , Cromatina/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunidade/genética , Masculino , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologiaRESUMO
Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.
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Cromatina/química , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Canal de Potássio KCNQ1/genética , Células Secretoras de Polipeptídeo Pancreático/metabolismo , Células Secretoras de Somatostatina/metabolismo , Glicemia/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Epigenômica , Jejum , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células Secretoras de Glucagon/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células Secretoras de Insulina/patologia , Canal de Potássio KCNQ1/metabolismo , Família Multigênica , Células Secretoras de Polipeptídeo Pancreático/patologia , Polimorfismo Genético , Análise de Célula Única , Células Secretoras de Somatostatina/patologia , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.
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Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/fisiologia , Homem de Neandertal/genética , Neurônios/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alelos , Processamento Alternativo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Evolução Biológica , Sistemas CRISPR-Cas , Proliferação de Células , Córtex Cerebral/citologia , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Genoma , Genoma Humano , Haplótipos , Hominidae/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Antígeno Neuro-Oncológico Ventral , Organoides , Sinapses/fisiologiaRESUMO
The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.
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Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Células A549 , Sítios de Ligação/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Mutação com Ganho de Função , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Polimerase II/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.
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Cromatina/química , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Animais , Linhagem da Célula/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Embrião de Mamíferos , Embrião não Mamífero , Desenvolvimento Embrionário , Células Endoteliais/citologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Ligação Proteica , Análise de Célula Única , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+ cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.
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Linhagem da Célula , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Bleomicina , Sistemas CRISPR-Cas/genética , Proliferação de Células , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/genética , Hiperóxia/patologia , Pulmão/metabolismo , Camundongos Transgênicos , Fibrose Pulmonar/patologia , Análise de Célula ÚnicaRESUMO
Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.