RESUMO
During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.
Assuntos
Organogênese , Fatores de Transcrição , Diferenciação Celular , Linfonodos , Tecido LinfoideRESUMO
Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.
Assuntos
Polaridade Celular/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Epigênese Genética/genética , Macrófagos/citologia , Fator de Transcrição STAT6/metabolismo , Ativação Transcricional/genética , Animais , Mapeamento Cromossômico , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Interleucina-4/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Domínios e Motivos de Interação entre Proteínas/genética , Fator de Transcrição STAT6/genética , Transcriptoma/genéticaRESUMO
Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.
Assuntos
Epigênese Genética/imunologia , Epigenômica/métodos , Regulação da Expressão Gênica/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , PPAR gama/imunologia , Animais , Linhagem Celular , Células Cultivadas , Interleucina-4/imunologia , Interleucina-4/farmacologia , Ligantes , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismoRESUMO
The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.
Assuntos
Interleucina-4/metabolismo , Macrófagos/metabolismo , Fator de Transcrição STAT6/metabolismo , Animais , Western Blotting , Linhagem Celular , Elementos Facilitadores Genéticos , Citometria de Fluxo , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Citometria de Varredura a Laser , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Piroptose/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Hedgehog/metabolismo , Prosencéfalo/metabolismo , Receptor Notch1/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Diferenciação Celular , Embrião de Mamíferos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Patched-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Transdução de SinaisRESUMO
Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or ß-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Regiões Promotoras Genéticas , Interferência de RNA , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Transcrição Gênica , Transfecção , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Specified intestinal epithelial cells reprogram and contribute to the regeneration and renewal of the epithelium upon injury. Mutations that deregulate such renewal processes may contribute to tumorigenesis. Using intestinal organoids, we show that concomitant activation of Notch signaling and ablation of p53 induce a highly proliferative and regenerative cell state, which is associated with increased levels of Yap and the histone methyltransferase Mll1. The induced signaling system orchestrates high proliferation, self-renewal, and niche-factor-independent growth, and elevates the trimethylation of histone 3 at lysine 4 (H3K4me3). We demonstrate that Yap and Mll1 are also elevated in patient-derived colorectal cancer (CRC) organoids and control growth and viability. Our data suggest that Notch activation and p53 ablation induce a signaling circuitry involving Yap and the epigenetic regulator Mll1, which locks cells in a proliferative and regenerative state that renders them susceptible for tumorigenesis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Mutação , Organoides/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Imunidade Inata , Interferons , SARS-CoV-2RESUMO
Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease aetiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease using the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of post-mortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabelling of the same tissues. Moreover, we analysed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in idiopathic Parkinson's disease midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signalling and immunomodulatory treatments in Parkinson's disease.
Assuntos
Doença de Parkinson , Neurônios Dopaminérgicos/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Glicoproteínas de Membrana/metabolismo , Mesencéfalo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismoRESUMO
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.
Assuntos
Sítios de Ligação Microbiológicos/genética , Linhagem da Célula/genética , Rastreamento de Células/métodos , Código de Barras de DNA Taxonômico/métodos , Células-Tronco Hematopoéticas/citologia , Recombinação Genética/genética , Análise de Célula Única/métodos , Animais , Células Clonais/citologia , Células Clonais/metabolismo , Embrião de Mamíferos/citologia , Células Eritroides/citologia , Células Eritroides/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Camundongos , Mosaicismo , Células Mieloides/citologia , Células Mieloides/metabolismoRESUMO
Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowell-based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single-nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage-specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments.
Assuntos
Arabidopsis/genética , Flores/genética , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Núcleo Celular/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos , Inflorescência/genética , RNA de Plantas , RNA Nuclear Pequeno , Reprodutibilidade dos Testes , Plântula/genéticaRESUMO
Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.
Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , RNA Viral/metabolismo , Ribonucleases/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Fibroblastos/metabolismo , Fibroblastos/virologia , Herpes Simples/genética , Herpes Simples/patologia , Herpes Simples/virologia , Humanos , Biossíntese de Proteínas , Proteoma , RNA Viral/genética , Ribonucleases/genética , Transcriptoma , Proteínas Virais/genéticaRESUMO
The transcription factor EB (TFEB) promotes protein degradation by the autophagy and lysosomal pathway (ALP) and overexpression of TFEB was suggested for the treatment of ALP-related diseases that often affect the heart. However, TFEB-mediated ALP induction may perturb cardiac stress response. We used adeno-associated viral vectors type 9 (AAV9) to overexpress TFEB (AAV9-Tfeb) or Luciferase-control (AAV9-Luc) in cardiomyocytes of 12-week-old male mice. Mice were subjected to transverse aortic constriction (TAC, 27G; AAV9-Luc: n = 9; AAV9-Tfeb: n = 14) or sham (AAV9-Luc: n = 9; AAV9-Tfeb: n = 9) surgery for 28 days. Heart morphology, echocardiography, gene expression, and protein levels were monitored. AAV9-Tfeb had no effect on cardiac structure and function in sham animals. TAC resulted in compensated left ventricular hypertrophy in AAV9-Luc mice. AAV9-Tfeb TAC mice showed a reduced LV ejection fraction and increased left ventricular diameters. Morphological, histological, and real-time PCR analyses showed increased heart weights, exaggerated fibrosis, and higher expression of stress markers and remodeling genes in AAV9-Tfeb TAC compared to AAV9-Luc TAC. RNA-sequencing, real-time PCR and Western Blot revealed a stronger ALP activation in the hearts of AAV9-Tfeb TAC mice. Cardiomyocyte-specific TFEB-overexpression promoted ALP gene expression during TAC, which was associated with heart failure. Treatment of ALP-related diseases by overexpression of TFEB warrants careful consideration.
Assuntos
Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Remodelação VentricularRESUMO
The omnigenic model of complex disease stipulates that the majority of the heritability will be explained by the effects of common variation on genes in the periphery of core disease pathways. Rare variant associations, expected to explain far less of the heritability, may be enriched in core disease genes and thus will be instrumental in the understanding of complex disease pathogenesis and their potential therapeutic targets. Here, using complementary whole-exome sequencing, high-density imputation, and in vitro cellular assays, we identify candidate core genes in the pathogenesis of systemic lupus erythematosus (SLE). Using extreme-phenotype sampling, we sequenced the exomes of 30 SLE parent-affected-offspring trios and identified 14 genes with missense de novo mutations (DNM), none of which are within the >80 SLE susceptibility loci implicated through genome-wide association studies. In a follow-up cohort of 10, 995 individuals of matched European ancestry, we imputed genotype data to the density of the combined UK10K-1000 genomes Phase III reference panel across the 14 candidate genes. Gene-level analyses indicate three functional candidates: DNMT3A, PRKCD, and C1QTNF4. We identify a burden of rare variants across PRKCD associated with SLE risk (P = 0.0028), and across DNMT3A associated with two severe disease prognosis sub-phenotypes (P = 0.0005 and P = 0.0033). We further characterise the TNF-dependent functions of the third candidate gene C1QTNF4 on NF-κB activation and apoptosis, which are inhibited by the p.His198Gln DNM. Our results identify three novel genes in SLE susceptibility and support extreme-phenotype sampling and DNM gene discovery to aid the search for core disease genes implicated through rare variation.
Assuntos
Lúpus Eritematoso Sistêmico/genética , Adulto , Autoanticorpos , Cromatografia em Gel , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Exoma/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Células HEK293 , Humanos , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteína Quinase C-delta/genética , Adulto JovemRESUMO
STAT1 gain-of-function (GOF) variants lead to defective Th17 cell development and chronic mucocutaneous candidiasis (CMC), but frequently also to autoimmunity. Stimulation of cells with STAT1 inducing cytokines like interferons (IFN) result in hyperphosphorylation and delayed dephosphorylation of GOF STAT1. However, the mechanism how the delayed dephosphorylation exactly causes the increased expression of STAT1-dependent genes, and how the intracellular signal transduction from cytokine receptors is affected, remains unknown. In this study we show that the circulating levels of IFN-α were not persistently elevated in STAT1 GOF patients. Nevertheless, the expression of interferon signature genes was evident even in the patient with low or undetectable serum IFN-α levels. Chromatin immunoprecipitation (ChIP) experiments revealed that the active chromatin mark trimethylation of lysine 4 of histone 3 (H3K4me3), was significantly enriched in areas associated with interferon-stimulated genes in STAT1 GOF cells in comparison to cells from healthy donors. This suggests that the chromatin binding of GOF STAT1 variant promotes epigenetic changes compatible with higher gene expression and elevated reactivity to type I interferons, and possibly predisposes for interferon-related autoimmunity. The results also suggest that epigenetic rewiring may be responsible for treatment failure of Janus kinase 1/2 (JAK1/2) inhibitors in certain patients.
Assuntos
Epigênese Genética , Mutação com Ganho de Função , Predisposição Genética para Doença , Interferons/metabolismo , Fator de Transcrição STAT1/genética , Candidíase Mucocutânea Crônica/etiologia , Candidíase Mucocutânea Crônica/metabolismo , Candidíase Mucocutânea Crônica/patologia , Estudos de Casos e Controles , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Humanos , Fosforilação , Ligação Proteica , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E2 (PGE2) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8+ T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8+ T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8+ T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE2 during DC maturation and reproducible with several responder T-cell lines. In conclusion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE2 seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Dinoprostona/uso terapêutico , Receptor 3 Toll-Like/imunologia , Movimento Celular , Apresentação Cruzada , Dinoprostona/farmacologia , HumanosRESUMO
Retinoid X receptor (RXR) is an obligate heterodimeric partner of several nuclear receptors (NRs), and as such a central component of NR signaling regulating the immune and metabolic phenotype of macrophages. Importantly, the binding motifs of RXR heterodimers are enriched in the tissue-selective open chromatin regions of resident macrophages, suggesting roles in subtype specification. Recent genome-wide studies revealed that RXR binds to thousands of sites in the genome, but the mechanistic details how the cistrome is established and serves ligand-induced transcriptional activity remained elusive. Here we show that IL-4-mediated macrophage plasticity results in a greatly extended RXR cistrome via both direct and indirect actions of the transcription factor STAT6. Activation of STAT6 leads to chromatin remodeling and RXR recruitment to de novo enhancers. In addition, STAT6 triggers a secondary transcription factor wave, including PPARγ. PPARγ appears to be indispensable for the development of RXR-bound de novo enhancers, whose activities can be modulated by the ligands of the PPARγ:RXR heterodimer conferring ligand selective cellular responses. Collectively, these data reveal the mechanisms leading to the dynamic extension of the RXR cistrome and identify the lipid-sensing enhancer sets responsible for the appearance of ligand-preferred gene signatures in alternatively polarized macrophages.
Assuntos
Interleucina-4/fisiologia , Macrófagos/metabolismo , PPAR gama/metabolismo , Receptores X de Retinoides/metabolismo , Fator de Transcrição STAT6/metabolismo , Animais , Células Cultivadas , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Redes Reguladoras de Genes , Ligantes , Macrófagos/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/metabolismo , Receptores X de Retinoides/genética , Transdução de SinaisRESUMO
BACKGROUND: The incidence of brain metastases in breast cancer (BCBM) patients is increasing. These patients have a very poor prognosis, and therefore, identification of blood-based biomarkers, such as circulating tumor cells (CTCs), and understanding the genomic heterogeneity could help to personalize treatment options. METHODS: Both EpCAM-dependent (CellSearch® System) and EpCAM-independent Ficoll-based density centrifugation methods were used to detect CTCs from 57 BCBM patients. DNA from individual CTCs and corresponding primary tumors and brain metastases were analyzed by next-generation sequencing (NGS) in order to evaluate copy number aberrations and single nucleotide variations (SNVs). RESULTS: CTCs were detected after EpCAM-dependent enrichment in 47.7% of the patients (≥ 5 CTCs/7.5 ml blood in 20.5%). The CTC count was associated with ERBB2 status (p = 0.029) of the primary tumor as well as with the prevalence of bone metastases (p = 0.021). EpCAM-independent enrichment revealed CTCs in 32.6% of the patients, especially among triple-negative breast cancer (TNBC) patients (70.0%). A positive CTC status after enrichment of either method was significantly associated with decreased overall survival time (p < 0.05). Combining the results of both enrichment methods, 63.6% of the patients were classified as CTC positive. In three patients, the matched tumor tissue and single CTCs were analyzed by NGS showing chromosomal aberrations with a high genomic clonality and mutations in pathways potentially important in brain metastasis formation. CONCLUSION: The detection of CTCs, regardless of the enrichment method, is of prognostic relevance in BCBM patients and in combination with molecular analysis of CTCs can help defining patients with higher risk of early relapse and suitability for targeted treatment.
Assuntos
Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/secundário , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Contagem de Células , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Humanos , Mutação , Células Neoplásicas Circulantes/metabolismo , Prognóstico , Análise de SobrevidaRESUMO
Molecular diagnosis of mitochondrial disorders is challenging because of extreme clinical and genetic heterogeneity. By exome sequencing, we identified three different bi-allelic truncating mutations in TANGO2 in three unrelated individuals with infancy-onset episodic metabolic crises characterized by encephalopathy, hypoglycemia, rhabdomyolysis, arrhythmias, and laboratory findings suggestive of a defect in mitochondrial fatty acid oxidation. Over the course of the disease, all individuals developed global brain atrophy with cognitive impairment and pyramidal signs. TANGO2 (transport and Golgi organization 2) encodes a protein with a putative function in redistribution of Golgi membranes into the endoplasmic reticulum in Drosophila and a mitochondrial localization has been confirmed in mice. Investigation of palmitate-dependent respiration in mutant fibroblasts showed evidence of a functional defect in mitochondrial ß-oxidation. Our results establish TANGO2 deficiency as a clinically recognizable cause of pediatric disease with multi-organ involvement.
Assuntos
Alelos , Arritmias Cardíacas/genética , Cardiomiopatias/genética , Mutação , Arritmias Cardíacas/diagnóstico , Cardiomiopatias/diagnóstico , Pré-Escolar , Exoma , Feminino , Humanos , Lactente , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , LinhagemRESUMO
Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.