ABSTRACT
DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.
Subject(s)
Cell Differentiation , Induced Pluripotent Stem Cells , Neural Stem Cells , Neurons , RNA Polymerase II , Humans , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Cell Differentiation/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Neurons/metabolism , Neurons/cytology , Promoter Regions, Genetic/geneticsABSTRACT
Mammalian genomes fold into tens of thousands of long-range loops, but their functional role and physiologic relevance remain poorly understood. Here, using human post-mitotic neurons with rare familial Alzheimer's disease (FAD) mutations, we identify hundreds of reproducibly dysregulated genes and thousands of miswired loops prior to amyloid accumulation and tau phosphorylation. Single loops do not predict expression changes; however, the severity and direction of change in mRNA levels and single-cell burst frequency strongly correlate with the number of FAD-gained or -lost promoter-enhancer loops. Classic architectural proteins CTCF and cohesin do not change occupancy in FAD-mutant neurons. Instead, we unexpectedly find TAATTA motifs amenable to binding by DLX homeodomain transcription factors and changing noncoding RNAPolII signal at FAD-dynamic promoter-enhancer loops. DLX1/5/6 mRNA levels are strongly upregulated in FAD-mutant neurons coincident with a shift in excitatory-to-inhibitory gene expression and miswiring of multi-loops connecting enhancers to neural subtype genes. DLX1 overexpression is sufficient for loop miswiring in wildtype neurons, including lost and gained loops at enhancers with tandem TAATTA arrays and singular TAATTA motifs, respectively. Our data uncover a genome structure-function relationship between multi-loop miswiring and dysregulated excitatory and inhibitory transcriptional programs during lineage commitment of human neurons homozygously-engineered with rare FAD mutations.
ABSTRACT
DNA is folded into higher-order structures that shape and are shaped by genome function. The role for long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNAPolII) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitors (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNAPolII initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops, promoter-promoter loops, or unlooped. Genes transitioning from repression to RNAPolII initiation exhibit slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNAPolII-mediated elongation during neural cell fate transitions.
ABSTRACT
Nearly half of the human genome is comprised of diverse repetitive sequences ranging from satellite repeats to retrotransposable elements. Such sequences are susceptible to stepwise expansions, duplications, inversions, and recombination events which can compromise genome function. In this review, we discuss the higher-order folding mechanisms of compartmentalization and loop extrusion and how they shape, and are shaped by, heterochromatin. Using primarily mammalian model systems, we contrast mechanisms governing H3K9me3-mediated heterochromatinization of the repetitive genome and highlight emerging links between repetitive elements and chromatin folding.
Subject(s)
Heterochromatin , Repetitive Sequences, Nucleic Acid , Animals , Genome, Human , Heterochromatin/genetics , Humans , Mammals , Repetitive Sequences, Nucleic Acid/geneticsABSTRACT
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
Subject(s)
Cell Cycle Proteins , Chromatin , Chromosomal Proteins, Non-Histone , Replication Origin , Cell Cycle Proteins/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Replication , Humans , Replication Origin/genetics , S Phase , CohesinsABSTRACT
Muscle regeneration is the result of the concerted action of multiple cell types driven by the temporarily controlled phenotype switches of infiltrating monocyte-derived macrophages. Pro-inflammatory macrophages transition into a phenotype that drives tissue repair through the production of effectors such as growth factors. This orchestrated sequence of regenerative inflammatory events, which we termed regeneration-promoting program (RPP), is essential for proper repair. However, it is not well understood how specialized repair-macrophage identity develops in the RPP at the transcriptional level and how induced macrophage-derived factors coordinate tissue repair. Gene expression kinetics-based clustering of blood circulating Ly6Chigh, infiltrating inflammatory Ly6Chigh, and reparative Ly6Clow macrophages, isolated from injured muscle, identified the TGF-ß superfamily member, GDF-15, as a component of the RPP. Myeloid GDF-15 is required for proper muscle regeneration following acute sterile injury, as revealed by gain- and loss-of-function studies. Mechanistically, GDF-15 acts both on proliferating myoblasts and on muscle-infiltrating myeloid cells. Epigenomic analyses of upstream regulators of Gdf15 expression identified that it is under the control of nuclear receptors RXR/PPARγ. Finally, immune single-cell RNA-seq profiling revealed that Gdf15 is coexpressed with other known muscle regeneration-associated growth factors, and their expression is limited to a unique subpopulation of repair-type macrophages (growth factor-expressing macrophages [GFEMs]).
Subject(s)
Gene Expression Profiling/methods , Growth Differentiation Factor 15/genetics , Inflammation/genetics , Intercellular Signaling Peptides and Proteins/genetics , Macrophages/metabolism , Regeneration/genetics , Animals , Cell Differentiation/genetics , Cells, Cultured , Growth Differentiation Factor 15/metabolism , Inflammation/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Muscle Cells/metabolism , Muscles/injuries , Muscles/metabolism , Muscles/physiopathology , Myeloid Cells/metabolism , RNA-Seq/methodsABSTRACT
The cardiac natriuretic peptides (NPs) are well established as regulators of blood pressure and fluid volume, but they also stimulate adipocyte lipolysis and control the gene program of nonshivering thermogenesis in brown adipose tissue. The NP "clearance" receptor C (NPRC) functions to clear NPs from the circulation via peptide internalization and degradation and thus is an important regulator of NP signaling and adipocyte metabolism. It is well known that the Nprc gene is highly expressed in adipose tissue and dynamically regulated upon nutrition and environmental changes. However, the molecular basis for how Nprc gene expression is regulated is still poorly understood. Here, we identified the nuclear receptor transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) as a transcriptional regulator of Nprc expression in mouse adipocytes. During 3T3-L1 adipocyte differentiation, levels of Nprc expression increase in parallel with PPARγ induction. Rosiglitazone, a classic PPARγ agonist, increases, whereas siRNA knockdown of PPARγ reduces, Nprc expression in 3T3-L1 adipocytes. By using chromosome conformation capture and luciferase reporter assays, we demonstrate that PPARγ controls Nprc gene expression in adipocytes through its long-range distal enhancers. Furthermore, the induction of Nprc expression in adipose tissue during high-fat diet feeding is found to be associated with increased PPARγ enhancer activity. Our findings define PPARγ as a mediator of adipocyte Nprc gene expression and establish a new connection between PPARγ and the control of adipocyte NP signaling in obesity.
Subject(s)
Adipose Tissue , Natriuretic Peptides , PPAR gamma , 3T3-L1 Cells , Adipocytes/metabolism , Adipogenesis , Animals , Lipolysis , Mice , Obesity/metabolism , Signal TransductionABSTRACT
Retinoid X receptor (RXR) plays a pivotal role as a transcriptional regulator and serves as an obligatory heterodimerization partner for at least 20 other nuclear receptors (NRs). Given a potentially limiting/sequestered pool of RXR and simultaneous expression of several RXR partners, we hypothesized that NRs compete for binding to RXR and that this competition is directed by specific agonist treatment. Here, we tested this hypothesis on three NRs: peroxisome proliferator-activated receptor gamma (PPARγ), vitamin D receptor (VDR), and retinoic acid receptor alpha (RARα). The evaluation of competition relied on a nuclear translocation assay applied in a three-color imaging model system by detecting changes in heterodimerization between RXRα and one of its partners (NR1) in the presence of another competing partner (NR2). Our results indicated dynamic competition between the NRs governed by two mechanisms. First, in the absence of agonist treatment, there is a hierarchy of affinities between RXRα and its partners in the following order: RARα > PPARγ > VDR. Second, upon agonist treatment, RXRα favors the liganded partner. We conclude that recruiting RXRα by the liganded NR not only facilitates a stimulus-specific cellular response but also might impede other NR pathways involving RXRα.
Subject(s)
PPAR gamma/metabolism , Protein Multimerization , Receptors, Calcitriol/metabolism , Retinoic Acid Receptor alpha/metabolism , Retinoid X Receptor alpha/metabolism , HEK293 Cells , Humans , PPAR gamma/genetics , Receptors, Calcitriol/genetics , Retinoic Acid Receptor alpha/genetics , Retinoid X Receptor alpha/geneticsABSTRACT
The concept of tissue-specific gene expression posits that lineage-determining transcription factors (LDTFs) determine the open chromatin profile of a cell via collaborative binding, providing molecular beacons to signal-dependent transcription factors (SDTFs). However, the guiding principles of LDTF binding, chromatin accessibility and enhancer activity have not yet been systematically evaluated. We sought to study these features of the macrophage genome by the combination of experimental (ChIP-seq, ATAC-seq and GRO-seq) and computational approaches. We show that Random Forest and Support Vector Regression machine learning methods can accurately predict chromatin accessibility using the binding patterns of the LDTF PU.1 and four other key TFs of macrophages (IRF8, JUNB, CEBPA and RUNX1). Any of these TFs alone were not sufficient to predict open chromatin, indicating that TF binding is widespread at closed or weakly opened chromatin regions. Analysis of the PU.1 cistrome revealed that two-thirds of PU.1 binding occurs at low accessible chromatin. We termed these sites labelled regulatory elements (LREs), which may represent a dormant state of a future enhancer and contribute to macrophage cellular plasticity. Collectively, our work demonstrates the existence of LREs occupied by various key TFs, regulating specific gene expression programs triggered by divergent macrophage polarizing stimuli.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Macrophages/metabolism , Regulatory Sequences, Nucleic Acid , Transcription Factors/metabolism , Animals , Cells, Cultured , Computational Biology , Gene Expression Regulation/physiology , Genome , Machine Learning , Mice , Mice, Inbred C57BL , Protein Binding/physiology , Staining and Labeling/methods , Transcriptional Activation/physiologyABSTRACT
Tissue regeneration is a highly coordinated process with sequential events including immune cell infiltration, clearance of damaged tissues, and immune-supported regrowth of the tissue. Aging has a well-documented negative impact on this process globally; however, whether changes in immune cells per se are contributing to the decline in the body's ability to regenerate tissues with aging is not clearly understood. Here, we set out to characterize the dynamics of macrophage infiltration and their functional contribution to muscle regeneration by comparing young and aged animals upon acute sterile injury. Injured muscle of old mice showed markedly elevated number of macrophages, with a predominance for Ly6Chigh pro-inflammatory macrophages and a lower ratio of the Ly6Clow repair macrophages. Of interest, a recently identified repair macrophage-derived cytokine, growth differentiation factor 3 (GDF3), was markedly downregulated in injured muscle of old relative to young mice. Supplementation of recombinant GDF3 in aged mice ameliorated the inefficient regenerative response. Together, these results uncover a deficiency in the quantity and quality of infiltrating macrophages during aging and suggest that in vivo administration of GDF3 could be an effective therapeutic approach.
Subject(s)
Aging/pathology , Growth Differentiation Factor 3/administration & dosage , Growth Differentiation Factor 3/pharmacology , Muscle, Skeletal/injuries , Muscle, Skeletal/physiopathology , Regeneration/drug effects , Acute Disease , Aging/drug effects , Animals , Cell Differentiation/drug effects , Kinetics , Male , Mice , Muscle, Skeletal/drug effects , Myoblasts/drug effects , Myoblasts/metabolism , Myoblasts/pathology , PhenotypeABSTRACT
Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration.SIGNIFICANCE STATEMENT Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.
Subject(s)
DNA Damage/physiology , Motor Neurons/enzymology , Protein-Arginine N-Methyltransferases/physiology , Aging/metabolism , Amino Acid Sequence , Animals , Arginine/analogs & derivatives , Arginine/metabolism , Cell Line , Cyclic AMP Response Element-Binding Protein/physiology , DNA Breaks, Double-Stranded , DNA Repair , Isometric Contraction , Male , Mice , Mice, Knockout , Mice, Transgenic , Muscle Cells/enzymology , Muscle Cells/physiology , Neuromuscular Junction/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/deficiency , Protein-Arginine N-Methyltransferases/genetics , RNA Interference , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins/metabolism , Reflex, Abnormal , Rotarod Performance Test , Spinal Cord/cytology , Spinal Cord/growth & developmentABSTRACT
The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.
Subject(s)
Interleukin-4/metabolism , Macrophages/metabolism , STAT6 Transcription Factor/metabolism , Animals , Blotting, Western , Cell Line , Enhancer Elements, Genetic , Flow Cytometry , Gene Expression Regulation , Inflammasomes/metabolism , Laser Scanning Cytometry , Lipopolysaccharides/pharmacology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Pyroptosis/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Retinoid X Receptors (RXRs) are unique and enigmatic members of the nuclear receptor (NR) family with extensive and complex biological functions in cellular differentiation. On the one hand, RXRs through permissive heterodimerization with other NRs are able to integrate multiple lipid signaling pathways and are believed to play a central role to coordinate the development of the central nervous system. On the other hand, RXRs may have heterodimer-independent functions as well. Therefore, a more RXR-centric analysis is warranted to identify its genomic binding sites and regulated gene networks, which are orchestrating the earliest events in neuronal differentiation. Recently developed genome-wide approaches allow systematic analyses of the RXR-driven neural differentiation. Here we applied next generation sequencing-based methodology to track the dynamic redistribution of the RXR cistrome along the path of embryonic stem cell to glutamatergic neuron differentiation. We identified Retinoic Acid Receptor (RAR) and Liver X Receptor (LXR) as dominant heterodimeric partners of RXR in these cellular stages. Our data presented here characterize the RAR:RXR and LXR:RXR-mediated transcriptional program in embryonic stem cells, neural progenitors and terminally differentiated neurons. Considering the growing evidence for dysregulated RXR-mediated signaling in neurodegenerative disorders, such as Alzheimer's Disease or Amyotrophic Lateral Sclerosis, the data presented here will be also a valuable resource for the field of neuro(patho)biology.
Subject(s)
Cell Lineage/genetics , Gene Expression Regulation , Neurogenesis/genetics , Protein Multimerization , Retinoid X Receptors/metabolism , Transcription, Genetic , Animals , Base Sequence , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Lineage/drug effects , Gene Expression Regulation/drug effects , Homeodomain Proteins/metabolism , Ligands , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/drug effects , Mouse Embryonic Stem Cells/metabolism , Neurogenesis/drug effects , Protein Binding/drug effects , Time Factors , Transcription, Genetic/drug effects , Transcriptome/drug effects , Transcriptome/genetics , Tretinoin/pharmacologyABSTRACT
Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.
Subject(s)
Imaging, Three-Dimensional , Nucleosomes/metabolism , Animals , Automation , Cell Line, Tumor , Doxorubicin/pharmacology , Ethidium/metabolism , Humans , Mice , Nucleosomes/drug effects , Salts/pharmacologyABSTRACT
Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or ß-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses.
Subject(s)
Cell Differentiation , Cell Lineage , Embryonic Stem Cells/metabolism , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/metabolism , Wnt Signaling Pathway , Animals , Binding Sites , Cell Differentiation/drug effects , Cellular Reprogramming , Embryonic Stem Cells/drug effects , Gene Expression Regulation , HEK293 Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Mice , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/drug effects , Promoter Regions, Genetic , RNA Interference , Retinoic Acid Receptor alpha/genetics , Retinoic Acid Receptor alpha/metabolism , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolism , Transcription, Genetic , Transfection , Tretinoin/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.
Subject(s)
Embryonic Stem Cells , Enhancer Elements, Genetic , Cell Differentiation , Chromatin Immunoprecipitation , TretinoinABSTRACT
Obesity and its comorbidity incidence have increased worldwide during the past 10 years. In consequence, researchers have drawn their attention to the understanding of adipocyte differentiation. Several cellular model systems have been established; however no efficient protocol could be developed so far to differentiate the pluripotent embryonic stem cells to adipocytes. In this chapter, we describe a detailed protocol that is optimized for mouse embryonic stem cells. The result of this differentiation is a homogenous adipocyte monolayer culture that can be used for several applications including developmental and pharmacological research.
Subject(s)
Adipocytes/cytology , Adipogenesis , Cell Culture Techniques/methods , Mouse Embryonic Stem Cells/cytology , Animals , Cell Line , MiceABSTRACT
BACKGROUND: Systems biology approaches have become indispensable tools in biomedical and basic research. These data integrating bioinformatic methods gained prominence after high-throughput technologies became available to investigate complex cellular processes, such as transcriptional regulation and protein-protein interactions, on a scale that had not been studied before. Immunology is one of the medical fields that systems biology impacted profoundly due to the plasticity of cell types involved and the accessibility of a wide range of experimental models. MATERIALS AND METHODS: In this review, we summarize the most important recent genomewide studies exploring the function of peroxisome proliferator-activated receptor γ in macrophages and dendritic cells. PPARγ ChIP-seq experiments were performed in adipocytes derived from embryonic stem cells to complement the existing data sets and to provide comparators to macrophage data. Finally, lists of regulated genes generated from such experiments were analysed with bioinformatics and system biology approaches. RESULTS: We show that genomewide studies utilizing high-throughput data acquisition methods made it possible to gain deeper insights into the role of PPARγ in these immune cell types. We also demonstrate that analysis and visualization of data using network-based approaches can be used to identify novel genes and functions regulated by the receptor. CONCLUSIONS: The example of PPARγ in macrophages and dendritic cells highlights the crucial importance of systems biology approaches in establishing novel cellular functions for long-known signaling pathways.