ABSTRACT
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a method to detect specific interactions between DNA and DNA-associated proteins. It is valuable for the characterization of the binding of transcription factors or co-regulators genome wide. Furthermore, it can be used for epigenetic profiling, chromatin accessibility assessment, and identification of regulatory elements. Compared to the more commonly used chromatin immunoprecipitation (ChIP), CUT&RUN has several advantages including an in situ approach as well as no need for sonication. However, the biggest advantage is the reduced cell amounts that are required for CUT&RUN, which makes it more attractive for experiments with limited cell numbers. In this chapter, we describe a reliable CUT&RUN protocol for macrophages that can be performed within 2 days and includes a library preparation so that the sample can be directly sequenced.
Subject(s)
Macrophages , Macrophages/metabolism , Animals , DNA/metabolism , DNA/genetics , Chromatin Immunoprecipitation/methods , Mice , Chromatin/metabolism , Chromatin/genetics , Humans , DNA-Binding Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Glucocorticoids are potent immunosuppressive drugs, but long-term treatment leads to severe side-effects. While there is a commonly accepted model for GR-mediated gene activation, the mechanism behind repression remains elusive. Understanding the molecular action of the glucocorticoid receptor (GR) mediated gene repression is the first step towards developing novel therapies. We devised an approach that combines multiple epigenetic assays with 3D chromatin data to find sequence patterns predicting gene expression change. We systematically tested> 100 models to evaluate the best way to integrate the data types and found that GR-bound regions hold most of the information needed to predict the polarity of Dex-induced transcriptional changes. We confirmed NF-κB motif family members as predictors for gene repression and identified STAT motifs as additional negative predictors.
ABSTRACT
In hepatocytes, peroxisome proliferator-activated receptor α (PPARα) orchestrates a genomic and metabolic response required for homeostasis during fasting. This includes the biosynthesis of ketone bodies and of fibroblast growth factor 21 (FGF21). Here we show that in the absence of adipose triglyceride lipase (ATGL) in adipocytes, ketone body and FGF21 production is impaired upon fasting. Liver gene expression analysis highlights a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. Adipose tissue lipolysis induced by activation of the ß3-adrenergic receptor also triggers such PPARα-dependent responses not only in the liver but also in brown adipose tissue (BAT). Intact PPARα activity in hepatocytes is required for the cross-talk between adipose tissues and the liver during fat mobilization.
Subject(s)
Lipolysis , PPAR alpha , Adipose Tissue/metabolism , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Hepatocytes/metabolism , Ketone Bodies/metabolism , Lipolysis/physiology , PPAR alpha/metabolismABSTRACT
Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to the brain repair process. However, molecular mechanisms underlying CNS disease-induced SVZ NSPC-redirected migration to the lesion area are poorly understood. Here, we show that genetic depletion of the p75 neurotrophin receptor (p75NTR-/-) in mice reduced SVZ NSPC migration towards the lesion area after cortical injury and that p75NTR-/- NSPCs failed to migrate upon BDNF stimulation in vitro. Cortical injury rapidly increased p75NTR abundance in SVZ NSPCs via bone morphogenetic protein (BMP) receptor signaling. SVZ-derived p75NTR-/- NSPCs revealed an altered cytoskeletal network- and small GTPase family-related gene and protein expression. In accordance, BMP-treated non-migrating p75NTR-/- NSPCs revealed an altered morphology and α-tubulin expression compared to BMP-treated migrating wild-type NSPCs. We propose that BMP-induced p75NTR abundance in NSPCs is a regulator of SVZ NSPC migration to the lesion area via regulation of the cytoskeleton following cortical injury.
Subject(s)
Neural Stem Cells , Stroke , Animals , Lateral Ventricles/metabolism , Mice , Neurogenesis , Receptor, Nerve Growth Factor/metabolismABSTRACT
Palmitoleic acid (C16:1n7) has been identified as a regulator of physiological cardiac hypertrophy. In the present study, we aimed to investigate the molecular pathways involved in C16:1n7 responses in primary murine cardiomyocytes (PCM) and a mouse model of isoproterenol (ISO)-induced cardiac damage. PCMs were stimulated with C16:1n7 or a vehicle. Afterwards, RNA sequencing was performed using an Illumina HiSeq sequencer. Confirmatory analysis was performed in PCMs and HL-1 cardiomyocytes. For an in vivo study, 129 sv mice were orally treated with a vehicle or C16:1n7 for 22 days. After 5 days of pre-treatment, the mice were injected with ISO (25 mg/kg/d s. c.) for 4 consecutive days. Cardiac phenotyping was performed using echocardiography. In total, 129 genes were differentially expressed in PCMs stimulated with C16:1n7, including Angiopoietin-like factor 4 (Angptl4) and Pyruvate Dehydrogenase Kinase 4 (Pdk4). Both Angptl4 and Pdk4 are proxisome proliferator-activated receptor α/δ (PPARα/δ) target genes. Our in vivo results indicated cardioprotective and anti-fibrotic effects of C16:1n7 application in mice. This was associated with the C16:1n7-dependent regulation of the cardiac PPAR-specific signaling pathways. In conclusion, our experiments demonstrated that C16:1n7 might have protective effects on cardiac fibrosis and inflammation. Our study may help to develop future lipid-based therapies for catecholamine-induced cardiac damage.
Subject(s)
Cardiomegaly/drug therapy , Cardiotonic Agents/pharmacology , Catecholamines/toxicity , Fatty Acids, Monounsaturated/pharmacology , Gene Expression Regulation/drug effects , PPAR alpha/metabolism , PPAR delta/metabolism , Animals , Cardiomegaly/chemically induced , Cardiomegaly/metabolism , Cardiomegaly/pathology , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , PPAR alpha/genetics , PPAR delta/geneticsABSTRACT
Quantifying differential genome occupancy by chromatin immunoprecipitation (ChIP) remains challenging due to variation in chromatin fragmentation, immunoprecipitation efficiencies, and intertube variability. In this protocol, we add heterologous spike-ins from Drosophila chromatin as an internal control to the mice chromatin before immunoprecipitation to normalize for technical variation in ChIP-qPCR or ChIP-seq. The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used. For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021).
Subject(s)
Chromatin Immunoprecipitation/methods , Proteins/metabolism , Animals , Binding Sites , Drosophila melanogaster , High-Throughput Nucleotide Sequencing/methods , Mice , Real-Time Polymerase Chain ReactionABSTRACT
Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR's protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis-regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.
Subject(s)
Enhancer Elements, Genetic/immunology , Macrophages/immunology , Multiprotein Complexes , RNA-Seq , Receptors, Glucocorticoid , Transcription, Genetic/immunology , Animals , Inflammation/genetics , Inflammation/immunology , Mice , Multiprotein Complexes/genetics , Multiprotein Complexes/immunology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/immunologyABSTRACT
Glucocorticoids are potent anti-inflammatory drugs; however, their molecular mode of action remains complex and elusive. They bind to the glucocorticoid receptor (GR), a nuclear receptor that controls gene expression in almost all tissues in a cell type-specific manner. While GR's transcriptional targets mediate beneficial reactions in immune cells, they also harbor the potential of adverse metabolic effects in other cell types such as hepatocytes. Here, we have profiled nascent transcription upon glucocorticoid stimulation in LPS-activated primary murine macrophages using 4sU-seq. We compared our results to publicly available nascent transcriptomics data from murine liver and bioinformatically identified non-coding RNAs transcribed from intergenic GR binding sites in a tissue-specific fashion. These tissue-specific enhancer RNAs (eRNAs) correlate with target gene expression, reflecting cell type-specific glucocorticoid responses. We further associate GR-mediated eRNA expression with changes in H3K27 acetylation and BRD4 recruitment in inflammatory macrophages upon glucocorticoid treatment. In summary, we propose a common mechanism by which GR-bound enhancers regulate target gene expression by changes in histone acetylation, BRD4 recruitment and eRNA expression. We argue that local eRNAs are potential therapeutic targets downstream of GR signaling which may modulate glucocorticoid response in a cell type-specific way.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Glucocorticoids/pharmacology , Macrophages/metabolism , RNA/genetics , Acetylation/drug effects , Animals , Binding Sites , Gene Expression Profiling , Gene Expression Regulation/drug effects , Histones/metabolism , Lysine/metabolism , Macrophages/drug effects , Male , Mice, Inbred C57BL , Nuclear Proteins/metabolism , Organ Specificity/drug effects , RNA/metabolism , Receptors, Glucocorticoid/metabolism , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.
Subject(s)
DNA-Binding Proteins/genetics , DNA/genetics , Inflammation/genetics , Receptors, Glucocorticoid/genetics , Animals , DNA/metabolism , Gene Expression Regulation/genetics , Glucocorticoids/genetics , Glucocorticoids/metabolism , Humans , Inflammation/pathology , Mice , Protein Interaction Domains and Motifs/genetics , RNA-Seq , Transcriptional Activation/geneticsABSTRACT
AIM: Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well-defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. METHODS: We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. RESULTS: We identified a new phosphorylation site on Myocardin-Related Transcription Factor B (MRTF-B), a co-activator of serum response factor (SRF). Phosphorylation of MRTF-B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos. In addition, high-intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF-target genes, and prevent increases in protein synthesis after exercise. CONCLUSION: Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise-induced protein synthesis and potentially muscle growth.
Subject(s)
Chromatin/chemistry , Muscle, Skeletal/metabolism , Physical Conditioning, Animal , Serum Response Factor , Signal Transduction , Transcription Factors/metabolism , Animals , Exercise , Humans , Male , Mice , Mice, Inbred C57BL , Protein Biosynthesis , Serum Response Factor/genetics , Serum Response Factor/metabolismABSTRACT
Glucocorticoids (GCs) are widely used immunomodulators. They regulate gene expression by binding and activating the Glucocorticoid Receptor (GR), but underlying transcriptional mechanisms remain enigmatic. This review summarizes recent findings identifyingspecific GR-bound DNA sequences whose configuration may affect transcriptional output. Additional factors affecting GR's anti-inflammatory actions, including different chromatin states such as DNAse hypersensitive regions and histone marks will be discussed, together with the relevant transcriptional co-regulators and promoter/enhancer features. Furthermore, the involvement of non-coding RNAs such as lncRNAs, miRNAs and eRNAs adds another level of regulation to the GR's transcriptional activity. Characterizing and understanding these multiple mechanisms will be crucial for developing more targeted immunomodulatory therapies with reduced adverse effects such as obesity, diabetes and osteoporosis.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Glucocorticoids/pharmacology , Animals , Genomics , Humans , RNA, Untranslated , Receptors, Glucocorticoid/genetics , Transcription, GeneticABSTRACT
Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) is a powerful tool to map context-dependent genome-wide binding of nuclear hormone receptors and their coregulators. This information can provide important mechanistic insight into where, when and how DNA-protein interactions are linked to target gene regulation. Here we describe a simple, yet reliable ChIP-seq method, including nuclear isolation from frozen tissue samples, cross-linking DNA-protein complexes, chromatin shearing, immunoprecipitation, and purification of ChIP DNA. We also include a standard ChIP-seq data analysis pipeline to elaborate and analyze raw single-end or paired-end sequencing data, including quality control steps, peak calling, annotation, and motif enrichment.
Subject(s)
Chromatin Immunoprecipitation/methods , High-Throughput Nucleotide Sequencing/methods , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , DNA/metabolism , Humans , Sequence Analysis, DNA/methodsABSTRACT
The mesothelial lining of the lung, the visceral pleura, and of the heart, the epicardium, derive from a common multipotent precursor tissue, the mesothelium of the embryonic thoracic cavity that also contributes to organ-specific mesenchymal cell types. Insight into mesothelial mobilization and differentiation has prevailedin the developing heart while the mesenchymal transition and fate of the visceral pleura are poorly understood. Here, we use the fact that the early mesothelium of both the lung and the heart expresses the transcription factor gene Wt1, to comparatively analyze mesothelial mobilization in the two organs by a genetic cre-loxP-based conditional approach. We show that epicardial cells are mobilized in a large number between E12.5 and E14.5, whereas pleural mobilization occurs only sporadically and variably in few regions of the lung in a temporally highly confined manner shortly after E12.5. Mesothelium-specific inactivation of unique pathway components using a Wt1creERT2 line excluded a requirement for canonical WNT, NOTCH, HH, TGFB, PDGFRA, and FGFR1/FGFR2 signaling in the mesenchymal transition of the visceral pleura but indicated a deleterious effect of activated WNT, NOTCH, and HH signaling on lung development. Epicardial mobilization was negatively impacted on by loss of HH, PDGFRA, FGFR1/2 signaling. Epicardial overactivation of WNT, NOTCH, and HH disturbed epicardial and myocardial integrity. We conclude that mesothelial mobilization in the developing lung and heart differs in timing, quantity and pathway dependency, indicating the organ specificity of the program.
Subject(s)
Epithelium/embryology , Heart/embryology , Lung/embryology , Animals , Cell Movement/genetics , Cell Movement/physiology , Epithelial-Mesenchymal Transition/genetics , Epithelial-Mesenchymal Transition/physiology , Epithelium/metabolism , Female , Gestational Age , Immunohistochemistry , Lung/metabolism , Male , Mice , Mice, Mutant Strains , Mice, Transgenic , Myocardium/metabolism , Pregnancy , Signal Transduction/genetics , WT1 Proteins/deficiency , WT1 Proteins/genetics , WT1 Proteins/metabolism , Wnt Signaling Pathway/geneticsABSTRACT
Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism. Validating BMAL1 and REV-ERBα targets using luciferase assays and in vivo rescue, we demonstrate how a major role of the muscle clock is to promote diurnal cycles of neutral lipid storage while coordinately inhibiting lipid and protein catabolism prior to awakening. This occurs by BMAL1-dependent activation of Dgat2 and REV-ERBα-dependent repression of major targets involved in lipid metabolism and protein turnover (MuRF-1, Atrogin-1). Accordingly, muscle-specific loss of BMAL1 is associated with metabolic inefficiency, impaired muscle triglyceride biosynthesis, and accumulation of bioactive lipids and amino acids. Taken together, our data provide a comprehensive overview of how genomic binding of BMAL1 and REV-ERBα is related to temporal changes in gene expression and metabolite fluctuations.
Subject(s)
ARNTL Transcription Factors/physiology , Circadian Clocks/physiology , Muscle, Skeletal/physiology , Amino Acids/metabolism , Amino Acids/physiology , Animals , CLOCK Proteins/genetics , Circadian Rhythm/genetics , Gene Expression , Homeostasis , Humans , Lipid Metabolism/physiology , Lipids , Mice , Mice, Knockout , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Alternative macrophage activation, which relies on mitochondrial oxidative metabolism, plays a central role in the resolution of inflammation and prevents atherosclerosis. Moreover, macrophages handle large amounts of cholesterol and triglycerides derived from the engulfed modified lipoproteins during atherosclerosis. Although several microRNAs regulate macrophage polarization, the role of the microRNA-generating enzyme Dicer in macrophage activation during atherosclerosis is unknown. METHODS: To evaluate the role of Dicer in atherosclerosis, Apoe-/- mice with or without macrophage-specific Dicer deletion were fed a high-fat diet for 12 weeks. Anti-argonaute 2 RNA immunoprecipitation chip and RNA deep sequencing combined with microRNA functional screening were performed in the Dicer wild-type and knockout bone marrow-derived macrophages to identify the individual microRNAs and the mRNA targets mediating the phenotypic effects of Dicer. The role of the identified individual microRNA and its target in atherosclerosis was determined by tail vein injection of the target site blockers in atherosclerotic Apoe-/- mice. RESULTS: We show that Dicer deletion in macrophages accelerated atherosclerosis in mice, along with enhanced inflammatory response and increased lipid accumulation in lesional macrophages. In vitro, alternative activation was limited whereas lipid-filled foam cell formation was exacerbated in Dicer-deficient macrophages as a result of impaired mitochondrial fatty acid oxidative metabolism. Rescue of microRNA (miR)-10a, let-7b, and miR-195a expression restored the oxidative metabolism in alternatively activated Dicer-deficient macrophages. Suppression of ligand-dependent nuclear receptor corepressor by miR-10a promoted fatty acid oxidation, which mediated the lipolytic and anti-inflammatory effect of Dicer. miR-10a expression was negatively correlated to the progression of atherosclerosis in humans. Blocking the interaction between ligand-dependent nuclear receptor corepressor and miR-10a by target site blockers aggravated atherosclerosis development in mice. CONCLUSIONS: Dicer plays an atheroprotective role by coordinately regulating the inflammatory response and lipid metabolism in macrophages through enhancing fatty acid-fueled mitochondrial respiration, suggesting that promoting Dicer/miR-10a-dependent metabolic reprogramming in macrophages has potential therapeutic implications to prevent atherosclerosis.
Subject(s)
Atherosclerosis/pathology , Macrophages/metabolism , Ribonuclease III/metabolism , Aged , Aged, 80 and over , Animals , Antagomirs/metabolism , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/metabolism , Bone Marrow Cells/cytology , Diet, High-Fat , Fatty Acids/chemistry , Female , Humans , Macrophages/cytology , Male , Mice , Mice, Knockout , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , MicroRNAs/metabolism , Mitochondria/metabolism , Nuclear Receptor Co-Repressor 2/chemistry , Nuclear Receptor Co-Repressor 2/metabolism , Oxidative Stress , Ribonuclease III/geneticsABSTRACT
During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).
Subject(s)
Alternative Splicing/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cerebral Cortex/embryology , Cyclin-Dependent Kinase Inhibitor p57/genetics , Neurons/cytology , Transcription Factor 3/metabolism , Animals , Base Sequence , Binding Sites/genetics , Cell Cycle/genetics , Cerebral Cortex/cytology , Chromatin/metabolism , Cyclin-Dependent Kinase Inhibitor p57/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , Mice, Inbred C57BL , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/metabolism , Protein Binding , Transcription Factor 3/deficiency , Transcription, GeneticABSTRACT
Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP), and RNA polymerase II (RNA Pol II) ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for â¼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD) and activation of the positive transcription elongation factor (pTEFb). Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation.
Subject(s)
Genome , Protein Biosynthesis , RNA Polymerase II/metabolism , RNA Splicing/genetics , T-Lymphocytes/metabolism , Transcription, Genetic , Animals , Computer Systems , Mice, Transgenic , Phosphorylation , Protein Domains , RNA Polymerase II/chemistryABSTRACT
The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.
Subject(s)
Epistasis, Genetic , Pericardium/embryology , Pericardium/metabolism , T-Box Domain Proteins/genetics , Alleles , Animals , Epithelial-Mesenchymal Transition/genetics , Gene Dosage , Gene Expression Regulation, Developmental , Humans , Mice , Phenotype , T-Box Domain Proteins/metabolismABSTRACT
Glucocorticoids (GCs), as ligands for the glucocorticoid receptor (GR), represent one of the most effective and frequently used classes of drugs for anti-inflammatory and immunosuppressive therapy. In addition, its role in physiological and pathophysiological processes makes the GR an important research target. The past decades have yielded a wealth of insight into the physiological and pharmacological effects of GCs. Today's era of next generation sequencing techniques is now beginning to elucidate the molecular and genomic circuits underlying GR's cell type-specific actions. This review focuses on the concepts and insights gained from recent studies in two of the most important tissues for GC action: the liver (mediating GR's metabolic effects) and macrophages (as the main target of anti-inflammatory GC therapy). We summarize results obtained from transgenic mouse models, molecular and genome-wide studies to illustrate GR's complex interactions with DNA, chromatin, co-regulators and other transcription factors. Characterizing the cell type-specific transcriptional complexes assembled around GR will pave the road for the development of new anti-inflammatory and metabolic therapies in the future.
Subject(s)
Inflammation/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Glucocorticoids/metabolism , Humans , Inflammation/genetics , Mice , Mice, Transgenic , Protein Binding , Receptors, Glucocorticoid/geneticsABSTRACT
Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.