RÉSUMÉ
Interactions between communities of the gut microbiome and with the host could affect the onset and progression of metabolic associated fatty liver disease (MAFLD), and can be useful as new diagnostic and prognostic biomarkers. In this study, we performed a multi-omics approach to unravel gut microbiome signatures from 32 biopsy-proven patients (10 simple steatosis -SS- and 22 steatohepatitis -SH-) and 19 healthy volunteers (HV). Human and microbial transcripts were differentially identified between groups (MAFLD vs. HV/SH vs. SS), and analyzed for weighted correlation networks together with previously detected metabolites from the same set of samples. We observed that expression of Desulfobacteraceae bacterium, methanogenic archaea, Mushu phage, opportunistic pathogenic fungi Fusarium proliferatum and Candida sorbophila, protozoa Blastocystis spp. and Fonticula alba were upregulated in MAFLD and SH. Desulfobacteraceae bacterium and Mushu phage were hub species in the onset of MAFLD, whereas the activity of Fonticula alba, Faecalibacterium prausnitzii, and Mushu phage act as key regulators of the progression to SH. A combination of clinical, metabolomic, and transcriptomic parameters showed the highest predictive capacity for MAFLD and SH (AUC = 0.96). In conclusion, faecal microbiome markers from several community members contribute to the switch in signatures characteristic of MAFLD and its progression towards SH.
Sujet(s)
Acyltransferases , Microbiome gastro-intestinal , Stéatose hépatique non alcoolique , Calcium-independent phospholipase A2 , Humains , Microbiome gastro-intestinal/génétique , Génotype , Métabolome , Transcriptome/génétique , Acyltransferases/génétique , Calcium-independent phospholipase A2/génétique , Stéatose hépatique non alcoolique/génétique , Stéatose hépatique non alcoolique/microbiologieRÉSUMÉ
Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFß in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFß and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.
Sujet(s)
Protocoles de polychimiothérapie antinéoplasique/pharmacologie , Fibroblastes associés au cancer/immunologie , Carcinomes/traitement médicamenteux , Interféron bêta/immunologie , Facteur de croissance transformant bêta/antagonistes et inhibiteurs , Animaux , Protocoles de polychimiothérapie antinéoplasique/usage thérapeutique , Fibroblastes associés au cancer/effets des médicaments et des substances chimiques , Carcinomes/immunologie , Carcinomes/anatomopathologie , Lignée cellulaire tumorale/transplantation , Plasticité cellulaire/effets des médicaments et des substances chimiques , Plasticité cellulaire/immunologie , Modèles animaux de maladie humaine , Synergie des médicaments , Femelle , Humains , Inhibiteurs de points de contrôle immunitaires/pharmacologie , Inhibiteurs de points de contrôle immunitaires/usage thérapeutique , Souris , Récepteur-1 de mort cellulaire programmée/antagonistes et inhibiteurs , Récepteur-1 de mort cellulaire programmée/immunologie , Cellules stromales/effets des médicaments et des substances chimiques , Cellules stromales/immunologie , Facteur de croissance transformant bêta/métabolisme , Microenvironnement tumoral/effets des médicaments et des substances chimiques , Microenvironnement tumoral/immunologieRÉSUMÉ
mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
Sujet(s)
Techniques d'amplification d'acides nucléiques/méthodes , ARN messager/métabolisme , Algorithmes , Sites de fixation , Système acellulaire , Amorces ADN/métabolisme , Test de retard de migration électrophorétique , Entropie , Escherichia coli/génétique , Escherichia coli/métabolisme , Protéines Escherichia coli/composition chimique , Protéines Escherichia coli/génétique , Protéines Escherichia coli/métabolisme , Modèles moléculaires , Conformation d'acide nucléique , Biosynthèse des protéines , Pliage de l'ARN , ARN messager/composition chimique , Protéines recombinantes/biosynthèse , Protéines recombinantes/composition chimique , Protéines recombinantes/isolement et purification , Ribosomes/composition chimique , Ribosomes/métabolisme , Régions non traduitesRÉSUMÉ
Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.
Sujet(s)
Vaccins contre le SIDA/administration et posologie , Anticorps antiviraux/biosynthèse , Lymphocytes B/effets des médicaments et des substances chimiques , Centre germinatif/effets des médicaments et des substances chimiques , Protéine d'enveloppe gp120 du VIH/administration et posologie , Vaccination/méthodes , Adjuvants immunologiques/administration et posologie , Animaux , Affinité des anticorps , Lymphocytes B/cytologie , Lymphocytes B/immunologie , Cellules CHO , Cricetulus , Calendrier d'administration des médicaments , Femelle , Centre germinatif/cytologie , Centre germinatif/immunologie , Cellules HEK293 , Protéine d'enveloppe gp120 du VIH/biosynthèse , Humains , Immunogénicité des vaccins , Pompes à perfusion implantables , Lipide A/administration et posologie , Lipide A/analogues et dérivés , Souris , Souris de lignée C57BL , Pression osmotique , Protéines de fusion recombinantes/administration et posologie , Protéines de fusion recombinantes/biosynthèse , Vaccination/instrumentationRÉSUMÉ
BACKGROUND & AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.
Sujet(s)
Génomique/méthodes , Foie/croissance et développement , Nanoparticules , Interférence par ARN , Protéines adaptatrices de la transduction du signal/physiologie , Animaux , Protéines du cycle cellulaire , Expression des gènes , Gènes nf2 , Facteur de croissance des hépatocytes/génétique , Hépatomégalie/étiologie , Foie/métabolisme , Souris , Souris de lignée C57BL , Taille d'organe , Phosphoprotéines/physiologie , Protein-Serine-Threonine Kinases/génétique , Protéines proto-oncogènes/génétique , Serine-threonine kinase-3 , Protéine p53 suppresseur de tumeur/physiologie , Protéines de signalisation YAPRÉSUMÉ
Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.
Sujet(s)
Hématopoïèse extramédullaire/immunologie , Cellules souches hématopoïétiques/immunologie , Macrophages/immunologie , Myélopoïèse/immunologie , Rate/immunologie , Molécule-1 d'adhérence des cellules vasculaires/immunologie , Animaux , Apolipoprotéines E/génétique , Apolipoprotéines E/immunologie , Athérosclérose/génétique , Athérosclérose/immunologie , Athérosclérose/anatomopathologie , Régulation de l'expression des gènes/génétique , Régulation de l'expression des gènes/immunologie , Hématopoïèse extramédullaire/génétique , Cellules souches hématopoïétiques/anatomopathologie , Macrophages/anatomopathologie , Souris , Souris knockout , Myélopoïèse/génétique , Infarctus du myocarde/génétique , Infarctus du myocarde/immunologie , Infarctus du myocarde/anatomopathologie , Nanoparticules , Plaque d'athérosclérose/génétique , Plaque d'athérosclérose/immunologie , Plaque d'athérosclérose/anatomopathologie , Interférence par ARN , Récepteur du facteur de stimulation des colonies de macrophages/génétique , Récepteur du facteur de stimulation des colonies de macrophages/immunologie , Lectine-1 de type Ig liant l'acide sialique/génétique , Lectine-1 de type Ig liant l'acide sialique/immunologie , Rate/anatomopathologie , Molécule-1 d'adhérence des cellules vasculaires/génétiqueRÉSUMÉ
Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.
Sujet(s)
Protéines Argonaute/métabolisme , Facteurs d'initiation eucaryotes/métabolisme , Fibroblastes/cytologie , Foie/métabolisme , ARN messager/génétique , Animaux , Protéines Argonaute/génétique , Cellules cultivées , Embryon de mammifère/cytologie , Femelle , Fibroblastes/métabolisme , Souris , Souris de lignée C57BL , Souris transgéniques , Cellules NIH 3T3 , Interférence par ARNRÉSUMÉ
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.
Sujet(s)
Cellules endothéliales/métabolisme , Nanoparticules/composition chimique , Polymères/composition chimique , Interférence par ARN , Petit ARN interférent/administration et posologie , Animaux , Lignée cellulaire , Humains , Souris , Nanoparticules/ultrastructure , Tumeurs/génétique , Tumeurs/thérapie , Petit ARN interférent/génétique , Petit ARN interférent/usage thérapeutiqueRÉSUMÉ
Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting ß1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.
Sujet(s)
Carcinome hépatocellulaire/génétique , Hépatocytes/métabolisme , Intégrines alpha/génétique , Chaines bêta des intégrines/génétique , Tumeurs expérimentales du foie/génétique , Nanoparticules , Protéines proto-oncogènes c-met/métabolisme , Petit ARN interférent , Animaux , Prolifération cellulaire , Évolution de la maladie , Régulation négative , Techniques de knock-down de gènes , SourisRÉSUMÉ
XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1-deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α-deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage, and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.
Sujet(s)
Endoribonucleases/métabolisme , Métabolisme lipidique/génétique , Lipides/sang , Protein-Serine-Threonine Kinases/métabolisme , ARN messager/métabolisme , Animaux , Apolipoprotéines E/déficit , Apolipoprotéines E/génétique , Apolipoprotéines E/métabolisme , Cholestérol/sang , Protéines de liaison à l'ADN/antagonistes et inhibiteurs , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Modèles animaux de maladie humaine , Endoribonucleases/antagonistes et inhibiteurs , Endoribonucleases/génétique , Stéatose hépatique/métabolisme , Stéatose hépatique/anatomopathologie , Analyse de profil d'expression de gènes , Lipogenèse , Souris , Souris knockout , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-Serine-Threonine Kinases/génétique , Interférence par ARN , Petit ARN interférent/métabolisme , Facteurs de transcription des facteurs régulateurs X , Facteurs de transcription/antagonistes et inhibiteurs , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Protéine-1 liant la boite XRÉSUMÉ
An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.
Sujet(s)
Endosomes/métabolisme , Lysosomes/métabolisme , Protéines G rab5/métabolisme , Animaux , Polarité de la cellule , Cellules cultivées , Endocytose , Techniques de knock-down de gènes , Hépatocytes/cytologie , Hépatocytes/métabolisme , Isoenzymes/biosynthèse , Isoenzymes/déficit , Isoenzymes/génétique , Isoenzymes/métabolisme , Lipoprotéines LDL/métabolisme , Foie/cytologie , Foie/enzymologie , Foie/métabolisme , Souris , Corps multivésiculaires/métabolisme , Spécificité d'organe , Biosynthèse des protéines , Interférence par ARN , ARN messager/analyse , ARN messager/génétique , Facteurs temps , Protéines du transport vésiculaire/métabolisme , Protéines G rab5/biosynthèse , Protéines G rab5/déficit , Protéines G rab5/génétiqueRÉSUMÉ
The mammalian stress sensor IRE1α plays a central role in the unfolded protein, or endoplasmic reticulum (ER), stress response by activating its downstream transcription factor XBP1 via an unconventional splicing mechanism. IRE1α can also induce the degradation of a subset of mRNAs in a process termed regulated IRE1-dependent decay (RIDD). Although diverse mRNA species can be degraded by IRE1α in vitro, the pathophysiological functions of RIDD are only beginning to be explored. Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in young adults in the United States and is primarily caused by CYP1A2-, CYP2E1-, and CYP3A4-driven conversion of APAP into hepatotoxic metabolites. We demonstrate here that genetic ablation of XBP1 results in constitutive IRE1α activation in the liver, leading to RIDD of Cyp1a2 and Cyp2e1 mRNAs, reduced JNK activation, and protection of mice from APAP-induced hepatotoxicity. A pharmacological ER stress inducer that activated IRE1α suppressed the expression of Cyp1a2 and Cyp2e1 in WT, but not IRE1α-deficient mouse liver, indicating the essential role of IRE1α in the down-regulation of these mRNAs upon ER stress. Our study reveals an unexpected function of RIDD in drug metabolism.
Sujet(s)
Acétaminophène/effets indésirables , Lésions hépatiques dues aux substances/métabolisme , Stress du réticulum endoplasmique/physiologie , Endoribonucleases/métabolisme , Activation enzymatique/physiologie , Régulation de l'expression des gènes/génétique , Protein-Serine-Threonine Kinases/métabolisme , Stabilité de l'ARN/génétique , Animaux , Technique de Western , Cytochrome P-450 CYP1A2/génétique , Cytochrome P-450 CYP1A2/métabolisme , Cytochrome P-450 CYP2E1/génétique , Cytochrome P-450 CYP2E1/métabolisme , Amorces ADN/génétique , Protéines de liaison à l'ADN/déficit , Stress du réticulum endoplasmique/génétique , Délétion de gène , Cellules HEK293 , Humains , Souris , Stabilité de l'ARN/physiologie , Petit ARN interférent/génétique , Réaction de polymérisation en chaine en temps réel , Facteurs de transcription des facteurs régulateurs X , Facteurs de transcription/déficit , Protéine-1 liant la boite XRÉSUMÉ
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
RÉSUMÉ
Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.
Sujet(s)
Cellules présentatrices d'antigène/métabolisme , Cations/composition chimique , Extinction de l'expression des gènes , Glyceraldehyde 3-phosphate dehydrogenases/antagonistes et inhibiteurs , Lipides/administration et posologie , Nanoparticules/administration et posologie , Petit ARN interférent/administration et posologie , Animaux , Technique de Western , Moelle osseuse , Cellules cultivées , Cellules dendritiques/cytologie , Cellules dendritiques/métabolisme , Endocytose , Cytométrie en flux , Glyceraldehyde 3-phosphate dehydrogenases/génétique , Glyceraldehyde 3-phosphate dehydrogenases/métabolisme , Hépatocytes/cytologie , Hépatocytes/métabolisme , Antigènes CD45/antagonistes et inhibiteurs , Antigènes CD45/génétique , Antigènes CD45/métabolisme , Liposomes , Foie/métabolisme , Macrophages/cytologie , Macrophages/métabolisme , Souris , Souris de lignée C57BL , Nanoparticules/composition chimique , Interférence par ARN , Petit ARN interférent/génétiqueRÉSUMÉ
Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/métabolisme , Cholestérol LDL/métabolisme , Chromosomes humains de la paire 1/génétique , Prédisposition génétique à une maladie/génétique , Polymorphisme de nucléotide simple/génétique , Protéines adaptatrices du transport vésiculaire/biosynthèse , Protéines adaptatrices du transport vésiculaire/déficit , Protéines adaptatrices du transport vésiculaire/génétique , Animaux , Séquence nucléotidique , Sites de fixation , Protéines liant les séquences stimulatrices de type CCAAT/métabolisme , Cellules cultivées , Cholestérol LDL/sang , Études de cohortes , Maladie des artères coronaires/sang , Maladie des artères coronaires/génétique , Europe/ethnologie , Régulation de l'expression des gènes , Techniques de knock-down de gènes , Étude d'association pangénomique , Haplotypes/génétique , Hépatocytes/métabolisme , Humains , Lipides/sang , Lipoprotéines VLDL/sang , Lipoprotéines VLDL/métabolisme , Foie/cytologie , Foie/métabolisme , Souris , Infarctus du myocarde/sang , Infarctus du myocarde/génétique , Phénotype , Transcription génétiqueRÉSUMÉ
BACKGROUND: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. METHODOLOGY/PRINCIPAL FINDINGS: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. CONCLUSION/SIGNIFICANCE: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibition following UV-induced DNA damage is correlated.
Sujet(s)
Caféine/pharmacologie , Altération de l'ADN , Facteur B d'élongation transcriptionnelle positive/métabolisme , Petites ribonucléoprotéines nucléaires/métabolisme , Transcription génétique/effets des médicaments et des substances chimiques , Transcription génétique/effets des radiations , Rayons ultraviolets , DNA polymerase II/composition chimique , DNA polymerase II/métabolisme , Cellules HeLa , Humains , Liaison aux protéines/effets des médicaments et des substances chimiques , Liaison aux protéines/effets des radiationsRÉSUMÉ
The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.
Sujet(s)
Analyse de profil d'expression de gènes/méthodes , Interprétation d'images assistée par ordinateur/méthodes , Microscopie de fluorescence/méthodes , Facteurs de transcription/métabolisme , Activation de la transcription/physiologieRÉSUMÉ
Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.