RESUMEN
Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.
Asunto(s)
Linfocitos B/inmunología , Complejo II de Transporte de Electrones/genética , Inflamación/metabolismo , Linfocitosis/inmunología , Mitocondrias/metabolismo , Mutación/genética , Antiinflamatorios/farmacología , Respiración de la Célula , Células Cultivadas , Fumaratos/metabolismo , Glucólisis , Humanos , Inflamación/genética , Interleucina-6/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Consumo de Oxígeno , Estudios Prospectivos , Transducción de Señal , Secuenciación del ExomaRESUMEN
Plasmacytoid dendritic cells (pDCs) are an immune subset devoted to the production of high amounts of type 1 interferons in response to viral infections. Whereas conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), pDCs have been shown to develop from both CDPs and common lymphoid progenitors. Here, we found that pDCs developed predominantly from IL-7R+ lymphoid progenitor cells. Expression of SiglecH and Ly6D defined pDC lineage commitment along the lymphoid branch. Transcriptional characterization of SiglecH+Ly6D+ precursors indicated that pDC development requires high expression of the transcription factor IRF8, whereas pDC identity relies on TCF4. RNA sequencing of IL-7R+ lymphoid and CDP-derived pDCs mirrored the heterogeneity of mature pDCs observed in single-cell analysis. Both mature pDC subsets are able to secrete type 1 interferons, but only myeloid-derived pDCs share with cDCs their ability to process and present antigen.
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Células Dendríticas/inmunología , Células Madre/inmunología , Animales , Linfocitos B/citología , Linaje de la Célula , Células Cultivadas , Células Dendríticas/citología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Factores Reguladores del Interferón/metabolismo , Lectinas/metabolismo , Masculino , Ratones , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina-7/metabolismo , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
To gain insight into the cellular readout of DNA methylation, we established a strategy for systematically profiling the genome-wide distribution of chromatin-interacting factors. This enabled us to create genomic maps for the methyl-CpG-binding domain (MBD) family of proteins, including disease-relevant mutants, deletions, and isoforms. In vivo binding of MBD proteins occurs predominantly as a linear function of local methylation density, requiring functional MBD domains and methyl-CPGs. This interaction directs specificity of MBD proteins to methylated, CpG-dense, and inactive regulatory regions. In contrast, binding to unmethylated sites varies between MBD proteins and is mediated via alternative domains or protein-protein interactions. Such targeting is exemplified by NuRD-complex-mediated tethering of MBD2 to a subset of unmethylated, active regulatory regions. Interestingly, MBD3 also occupies these sites, but like MBD2, binding is independent of the presence of hydroxymethylation. These functional binding maps reveal methylation-dependent and -independent binding modes and revise current models of DNA methylation readout through MBD proteins.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Animales , Biotina/metabolismo , Cromatina/metabolismo , Islas de CpG , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células Madre Embrionarias , Estudio de Asociación del Genoma Completo , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Metastatic colonization of distant organs accounts for over 90% of deaths related to solid cancers, yet the molecular determinants of metastasis remain poorly understood. Here, we unveil a mechanism of colonization in the aggressive basal-like subtype of breast cancer that is driven by the NAD+ metabolic enzyme nicotinamide N-methyltransferase (NNMT). We demonstrate that NNMT imprints a basal genetic program into cancer cells, enhancing their plasticity. In line, NNMT expression is associated with poor clinical outcomes in patients with breast cancer. Accordingly, ablation of NNMT dramatically suppresses metastasis formation in pre-clinical mouse models. Mechanistically, NNMT depletion results in a methyl overflow that increases histone H3K9 trimethylation (H3K9me3) and DNA methylation at the promoters of PR/SET Domain-5 (PRDM5) and extracellular matrix-related genes. PRDM5 emerged in this study as a pro-metastatic gene acting via induction of cancer-cell intrinsic transcription of collagens. Depletion of PRDM5 in tumor cells decreases COL1A1 deposition and impairs metastatic colonization of the lungs. These findings reveal a critical activity of the NNMT-PRDM5-COL1A1 axis for cancer cell plasticity and metastasis in basal-like breast cancer.
Asunto(s)
Neoplasias , Nicotinamida N-Metiltransferasa , Animales , Ratones , Nicotinamida N-Metiltransferasa/genética , Nicotinamida N-Metiltransferasa/metabolismo , Neoplasias/metabolismo , Metilación de ADN , Epigénesis GenéticaRESUMEN
The NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver-derived EBs. However, NFIA-ETO2-expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.
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Leucemia Eritroblástica Aguda , Proteínas Represoras , Animales , Ratones , Proteínas Represoras/genética , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Diferenciación Celular/genética , Eritroblastos/metabolismo , Factores de Transcripción NFI/metabolismoRESUMEN
The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.
Asunto(s)
Neoplasias de la Mama , ARN Largo no Codificante , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Desmosomas/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Invasividad Neoplásica/genética , ARN Largo no Codificante/genética , Factores de Transcripción SOXCRESUMEN
OBJECTIVE: Mucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory and antimicrobial functions and are implicated in liver fibrogenesis. It is not well understood which liver cells function as antigen (Ag)-presenting cells for MAIT cells, and under which conditions stimulatory Ags reach the circulation. DESIGN: We used different types of primary human liver cells in Ag-presentation assays to blood-derived and liver-derived MAIT cells. We assessed MAIT cell stimulatory potential of serum from healthy subjects and patients with portal hypertension undergoing transjugular intrahepatic portosystemic shunt stent, and patients with inflammatory bowel disease (IBD). RESULTS: MAIT cells were dispersed throughout healthy human liver and all tested liver cell types stimulated MAIT cells, hepatocytes being most efficient. MAIT cell activation by liver cells occurred in response to bacterial lysate and pure Ag, and was prevented by non-activating MR1 ligands. Serum derived from peripheral and portal blood, and from patients with IBD stimulated MAIT cells in MR1-dependent manner. CONCLUSION: Our findings reveal previously unrecognised roles of liver cells in Ag metabolism and activation of MAIT cells, repression of which creates an opportunity to design antifibrotic therapies. The presence of MAIT cell stimulatory Ags in serum rationalises the observed activated MAIT cell phenotype in liver. Increased serum levels of gut-derived MAIT cell stimulatory ligands in patients with impaired intestinal barrier function indicate that intrahepatic Ag-presentation may represent an important step in the development of liver disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Células T Invariantes Asociadas a Mucosa , Humanos , Antígenos de Histocompatibilidad Menor , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Activación de LinfocitosRESUMEN
Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Memoria Inmunológica , Receptores de Antígenos de Linfocitos T/análisis , Animales , Homeostasis , RatonesRESUMEN
The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.
Asunto(s)
Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220+CD117intCD19-NK1.1- uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among them, the Ly6D+SiglecH-CD11c- fraction was lymphoid-restricted exhibiting strong B-cell potential, whereas the Ly6D-SiglecH-CD11c- fraction showed mixed lympho-myeloid potential. Single-cell RNA sequencing of these subsets revealed that the latter population comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D+SiglecH-CD11c- Subsequent functional assays confirmed that B220+CD117intCD19-NK1.1- single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D+SiglecH-CD11c- subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220+CD117intCD19-NK1.1- progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Análisis de Secuencia de ARN/métodos , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Diferenciación Celular , Linaje de la Célula , Femenino , Perfilación de la Expresión Génica , Heterogeneidad Genética , Células Madre Hematopoyéticas/citología , Secuenciación de Nucleótidos de Alto Rendimiento , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/fisiología , Masculino , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/fisiología , Análisis de la Célula IndividualRESUMEN
The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.
Asunto(s)
Evolución Biológica , Extremidades/anatomía & histología , Extremidades/embriología , Proteínas Hedgehog/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Tipificación del Cuerpo , Bovinos , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Esbozos de los Miembros/anatomía & histología , Esbozos de los Miembros/embriología , Masculino , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.
Asunto(s)
Citosina/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Epigenómica , Animales , Diferenciación Celular , Islas de CpG , Células Madre Embrionarias/citología , Ratones , Neuronas/citología , Regiones Promotoras Genéticas/genética , Unión Proteica , Células Madre/citología , Factores de Transcripción/metabolismoRESUMEN
The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/biosíntesis , Transición Epitelial-Mesenquimal/fisiología , Fosfoproteínas/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Zixina/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Fosfoproteínas/genética , Transducción de Señal , Factores de Transcripción de Dominio TEA , Transactivadores , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Señalizadoras YAP , Zixina/genéticaRESUMEN
Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.
Asunto(s)
Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Transcripción Genética , 5-Metilcitosina/análogos & derivados , Animales , Sitios de Unión , Factor de Unión a CCCTC , Diferenciación Celular , Islas de CpG/genética , Citosina/análogos & derivados , Citosina/farmacología , Células Madre Embrionarias , Regulación de la Expresión Génica/efectos de los fármacos , Genoma , Ratones , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genéticaRESUMEN
Tumor growth depends on the formation of new blood vessels (tumor angiogenesis) either from preexisting vessels or by the recruitment of bone marrow-derived cells. Despite encouraging results obtained with preclinical cancer models, the therapeutic targeting of tumor angiogenesis has thus far failed to deliver an enduring clinical response in cancer patients. One major obstacle for improving anti-angiogenic therapy is the lack of validated biomarkers, which allow patient stratification for suitable treatment and a rapid assessment of therapy response. Toward these goals, we have employed several mouse models of tumor angiogenesis to identify cell populations circulating in their blood that correlated with the extent of tumor angiogenesis and therapy response. Flow cytometry analyses of different combinations of cell surface markers that define subsets of bone marrow-derived cells were performed on peripheral blood mononuclear cells from tumor-bearing and healthy mice. We identified one cell population, CD45(dim)VEGFR1(-)CD31(low), that was increased in levels during active tumor angiogenesis in a variety of transgenic and syngeneic transplantation mouse models of cancer. Treatment with various anti-angiogenic drugs did not affect CD45(dim)VEGFR1(-)CD31(low) cells in healthy mice, whereas in tumor-bearing mice, a consistent reduction in their levels was observed. Gene expression profiling of CD45(dim)VEGFR1(-)CD31(low) cells characterized these cells as an immature B cell population. These immature B cells were then directly validated as surrogate marker for tumor angiogenesis and of pharmacologic responses to anti-angiogenic therapies in various mouse models of cancer.
Asunto(s)
Linfocitos B/inmunología , Biomarcadores/metabolismo , Neoplasias/patología , Neovascularización Patológica , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Membrana Celular/metabolismo , Técnicas de Cocultivo , Biología Computacional , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Antígenos Comunes de Leucocito/metabolismo , Leucocitos Mononucleares/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cellular differentiation of the T-cell branch of the immune system begins with the HSC, which undergoes a series of stages characterized by progressive restriction in multipotency and acquisition of specific lineage identity At the molecular level, the restriction of cell potential, commitment, and differentiation to a specific lineage is achieved through the coordinated control of gene expression and epigenetic mechanisms. Here, we analyzed and compared the gene expression profiles and the genome-wide histone modification marks H3K4me3 (H3 lysine 4 trimethylation) and H3K27me3 (H3 lysine 27 trimethylation) in (i) in vitro propagated HSCs, (ii) in vitro generated and propagated pro-T cells derived from these stem cells, and (iii) double-positive thymocytes derived from these pro-T cells after injection into Rag-deficient mice. The combined analyses of the different datasets in this unique experimental system highlighted the importance of both transcriptional and epigenetic repression in shaping the early phases of T-cell development.
Asunto(s)
Epigénesis Genética , Epigenómica/métodos , Células Madre Multipotentes/metabolismo , Linfocitos T/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Análisis por Conglomerados , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Multipotentes/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Precursoras de Linfocitos T/citología , Células Precursoras de Linfocitos T/metabolismo , Linfocitos T/citologíaRESUMEN
Development of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEC(high)) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire(+)mTEC(high) to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire(+)mTEC(high) cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire(+)mTEC(high), with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.
Asunto(s)
Antígenos/genética , Células Epiteliales/inmunología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Timo/metabolismo , Enfermedad Aguda , Animales , Antígenos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Femenino , Expresión Génica/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Timo/patologíaRESUMEN
Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.
Asunto(s)
Genes Dominantes/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas de la Membrana/genética , Mutación/genética , Lipofuscinosis Ceroideas Neuronales/epidemiología , Lipofuscinosis Ceroideas Neuronales/genética , Adulto , Edad de Inicio , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Segregación Cromosómica/genética , Exones/genética , Familia , Femenino , Dosificación de Gen/genética , Regulación de la Expresión Génica , Ligamiento Genético , Humanos , Lipoilación , Lisosomas/metabolismo , Lisosomas/ultraestructura , Masculino , Datos de Secuencia Molecular , Lipofuscinosis Ceroideas Neuronales/patología , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Linaje , Transporte de Proteínas , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Modelos Animales de Enfermedad , Músculo Esquelético , Distrofia Miotónica , Unión Neuromuscular , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/fisiopatología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Unión Neuromuscular/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/inervación , Músculo Esquelético/patología , Ratones , Humanos , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Receptores Colinérgicos/metabolismo , Receptores Colinérgicos/genética , Masculino , Ratones Endogámicos C57BLRESUMEN
After infection of B cells, Epstein-Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates nicotinamide adenine dinucleotide (NAD) de novo biosynthesis by driving expression of the metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match adenosine triphosphate (ATP) production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is therefore a druggable metabolic vulnerability of EBV-driven B cell transformation, opening therapeutic possibilities for EBV-related diseases.