ABSTRACT
Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.
Subject(s)
Atlases as Topic , Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Molecular Sequence Annotation , Organ Specificity , Cell Line , Cells, Cultured , Cluster Analysis , Genetic Predisposition to Disease/genetics , HeLa Cells , Humans , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription Initiation Site , Transcription Initiation, GeneticABSTRACT
Cellular differentiation is orchestrated by lineage-specific transcription factors and associated with cell type-specific epigenetic signatures. In the present study, we used stage-specific, epigenetic "fingerprints" to deduce key transcriptional regulators of the human monocytic differentiation process. We globally mapped the distribution of epigenetic enhancer marks (histone H3 lysine 4 monomethylation, histone H3 lysine 27 acetylation, and the histone variant H2AZ), describe general properties of marked regions, and show that cell type-specific epigenetic "fingerprints" are correlated with specific, de novo-derived motif signatures at all of the differentiation stages studied (ie, hematopoietic stem cells, monocytes, and macrophages). We validated the novel, de novo-derived, macrophage-specific enhancer signature, which included ETS, CEBP, bZIP, EGR, E-Box and NF-κB motifs, by ChIP sequencing for a subset of motif corresponding transcription factors (PU.1, C/EBPß, and EGR2), confirming their association with differentiation-associated epigenetic changes. We describe herein the dynamic enhancer landscape of human macrophage differentiation, highlight the power of genome-wide epigenetic profiling studies to reveal novel functional insights, and provide a unique resource for macrophage biologists.
Subject(s)
Cell Differentiation/genetics , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic , Monocytes/cytology , Monocytes/metabolism , Transcription Factors/metabolism , Acetylation , Base Sequence , CCAAT-Enhancer-Binding Protein-beta/metabolism , Disease/genetics , Early Growth Response Protein 2/metabolism , Genetic Variation , Histones/metabolism , Humans , Macrophages/cytology , Macrophages/metabolism , Methylation , Models, Biological , Molecular Sequence Data , Nucleotide Motifs/genetics , Organ Specificity/genetics , Protein Binding , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolismABSTRACT
Malignant transformation is frequently associated with disease-specific epigenetic alterations, but the underlying mechanisms and pathophysiological consequences remain poorly understood. Here, we used global comparative DNA methylation profiling at CG-rich regions of 27 acute myeloid leukemia (AML) samples to select a subset of aberrantly methylated CG-rich regions (~400 regions, ~15,000 CpGs) for quantitative DNA methylation profiling in a large cohort of AML patients (n = 196) using MALDI-TOF analysis of bisulfite-treated DNA. Meta-analysis separated a subgroup of CG-rich regions showing highly correlated DNA methylation changes that were marked by histone H3 lysine 27 trimethylation in normal hematopoietic progenitor cells. While the group of non-polycomb group (PcG) target regions displayed methylation patterns that correlated well with molecular and cytogenetic markers, PcG target regions displayed a much weaker association with genetic features. However, the degree of methylation gain across the latter panel showed significant correlation with active DNMT3A levels and with overall survival. Our study suggests that both epigenetic as well as genetic aberrations underlay AML-related changes in DNA methylation at CG-rich regions and that the former may provide a marker to improve classification and prognostication of adult AML patients.
Subject(s)
Biomarkers, Tumor/genetics , Cell Transformation, Neoplastic/pathology , CpG Islands , DNA Methylation , Epigenesis, Genetic , Hematopoietic Stem Cells/pathology , Leukemia, Myeloid, Acute/genetics , Adult , Case-Control Studies , Cell Transformation, Neoplastic/genetics , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Prognosis , Promoter Regions, Genetic , Survival Rate , Young AdultABSTRACT
Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. We have developed a novel technique for detecting CpG-methylated DNA termed methyl-binding (MB)-PCR. This technique utilizes a recombinant protein with high affinity for CpG-methylated DNA that is coated onto the walls of a PCR vessel and selectively captures methylated DNA fragments from a mixture of genomic DNA. The retention and, hence, the degree of methylation of a specific DNA fragment (e.g. a CpG island promoter of a specific gene) is detected in the same tube by gene-specific PCR. MB-PCR does not require bisulfite treatment or methylation-sensitive restriction and provides a quick, simple and extremely sensitive technique allowing the detection of methylated DNA, in particular in tumor tissue or tumor cells from limited samples. Using this novel approach, we determined the methylation status of several established and candidate tumor suppressor genes and identified the ICSBP gene, encoding the myeloid and B-cell-specific transcription factor interferon consensus sequence-binding protein, as a target for aberrant hypermethylation in acute myeloid leukemia.
Subject(s)
CpG Islands , DNA Methylation , DNA, Neoplasm/analysis , DNA-Binding Proteins , Leukemia, Myeloid/genetics , Polymerase Chain Reaction/methods , Acute Disease , Cell Line, Tumor , Cells, Cultured , DNA, Neoplasm/chemistry , Genes, Tumor Suppressor , Humans , Interferon Regulatory Factors/genetics , Promoter Regions, Genetic , Time Factors , Tumor Cells, CulturedABSTRACT
The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies.
Subject(s)
CpG Islands/genetics , DNA Methylation , Leukemia, Myeloid/genetics , Acute Disease , Cell Line, Tumor , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Gene Expression Profiling , Genome, Human , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/immunology , Immunoprecipitation/methods , Leukemia, Myeloid/metabolism , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Reproducibility of Results , U937 CellsABSTRACT
Interleukin (IL)-4 is a central regulator of T helper 2 (Th2) immune responses, and also has a major impact on innate immune cells. This cytokine primes macrophages for immune responses to parasites and induces a distinct macrophage phenotype that may also promote tissue repair. IL-4 signaling in macrophages is primarily mediated by the transcription factor signal transducer and activator of transcription 6 (Stat6), which in turn regulates a number of secondary DNA binding proteins that may participate in shaping the resulting phenotype. The impact of secondary transcription factors on IL-4-treated macrophages, however, is largely unknown. Here we show that interferon regulatory factor 4 (Irf4) is strongly induced on RNA and protein level in bone marrow-derived macrophages upon priming with IL-4. Using microarray-based whole genome expression analysis, we also demonstrate that a subset of IL-4 regulated genes, including several MHC-II genes, Ciita, Cyp1b1, and Il1rn, are dysregulated in Irf4-deficient macrophages. The presented data suggests a non-redundant role for Irf4 in shaping the phenotype of alternatively primed macrophages.
Subject(s)
Interferon Regulatory Factors/metabolism , Macrophages/metabolism , STAT6 Transcription Factor/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP1B1 , Gene Expression Regulation , Immunity, Innate/genetics , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-4/immunology , Interleukin-4/metabolism , Macrophage Activation/genetics , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/immunology , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
BACKGROUND: In mammals, the dynamics of DNA methylation, in particular the regulated, active removal of cytosine methylation, has remained a mystery, partly due to the lack of appropriate model systems to study DNA demethylation. Previous work has largely focused on proliferating cell types that are mitotically arrested using pharmacological inhibitors to distinguish between active and passive mechanisms of DNA demethylation. RESULTS: We explored this epigenetic phenomenon in a natural setting of post-mitotic cells: the differentiation of human peripheral blood monocytes into macrophages or dendritic cells, which proceeds without cell division. Using a global, comparative CpG methylation profiling approach, we identified many novel examples of active DNA demethylation and characterized accompanying transcriptional and epigenetic events at these sites during monocytic differentiation. We show that active DNA demethylation is not restricted to proximal promoters and that the time-course of demethylation varies for individual CpGs. Irrespective of their location, the removal of methylated cytosines always coincided with the appearance of activating histone marks. CONCLUSIONS: Demethylation events are highly reproducible in monocyte-derived dendritic cells from different individuals. Our data suggest that active DNA demethylation is a precisely targeted event that parallels or follows the modification of histones, but is not necessarily coupled to alterations in transcriptional activity.
Subject(s)
DNA Methylation/genetics , Histones/metabolism , Mitosis/genetics , Monocytes/cytology , Monocytes/metabolism , Protein Processing, Post-Translational/genetics , Transcription, Genetic , Cell Differentiation , Cell Proliferation , Dendritic Cells/cytology , Dendritic Cells/metabolism , Gene Expression Profiling , Gene Expression Regulation , Histones/genetics , Humans , Macrophages/cytology , Macrophages/metabolism , Models, Genetic , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Aberrant DNA methylation at CpG islands is thought to contribute to cancer initiation and progression, but mechanisms that establish and maintain DNA methylation status during tumorigenesis or normal development remain poorly understood. In this study, we used methyl-CpG immunoprecipitation to generate comparative DNA methylation profiles of healthy and malignant cells (acute leukemia and colorectal carcinoma) for human CpG islands across the genome. While searching for sequence patterns that characterize DNA methylation states, we discovered several nonredundant sequences in CpG islands that were resistant to aberrant de novo methylation in cancer and that resembled consensus binding sites for general transcription factors (TF). Comparing methylation profiles with global CpG island binding data for specific protein 1, nuclear respiratory factor 1, and yin-yang 1 revealed that their DNA binding activity in normal blood cells correlated strictly with an absence of de novo methylation in cancer. In addition, global evidence showed that binding of any of these TFs to their consensus motif depended on their co-occurrence with neighboring consensus motifs. In summary, our results had two major implications. First, they pointed to a major role for cooperative binding of TFs in maintaining the unmethylated status of CpG islands in health and disease. Second, our results suggest that the majority of de novo methylated CpG islands are characterized by the lack of sequence motif combinations and the absence of activating TF binding.
Subject(s)
CpG Islands , DNA Methylation/genetics , Neoplasms/genetics , Neoplasms/metabolism , Transcription Factors/metabolism , Adult , Base Sequence , Gene Expression Profiling , Humans , Leukemia/genetics , Male , Middle Aged , Models, Biological , Oligonucleotide Array Sequence Analysis , Protein Binding , Tumor Cells, Cultured , U937 Cells , Young AdultABSTRACT
Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/physiology , Cell Differentiation/physiology , Gene Expression Regulation/physiology , Macrophages/cytology , Macrophages/physiology , Monocytes/physiology , CCAAT-Enhancer-Binding Protein-beta/genetics , Cell Separation , Humans , Leukapheresis , Monocytes/cytology , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA/genetics , RNA/isolation & purification , Transcription, Genetic , TransfectionABSTRACT
This report investigates the molecular basis for tissue-restricted and regulated expression of the pattern recognition receptor Toll-like receptor (TLR)2 in human monocytes and macrophages. To define the proximal promoter, the full 5'-sequence and transcriptional start sites of TLR2 mRNA were determined. The human TLR2 gene was found to consist of two 5' noncoding exons followed by a third coding exon. Alternative splicing of exon II was detected primarily in human blood monocytes. The proximal promoter, exon I, and part of intron I were found to be located in a CpG island. Although CpG methylation of the proximal human TLR2 promoter in cell lines correlated with TLR2 repression, the promoter was unmethylated in primary cells, indicating that CpG methylation does not contribute to the cell-type specific expression of human TLR2 in normal tissues. The promoter sequence contains putative binding sites for several transcription factors, including Sp1 and Ets family members. Reporter gene analysis revealed a minimal promoter of 220 bp that was found to be regulated by Sp1, Sp3, and possibly PU.1. Interestingly, no sequence homology was detected between human and murine TLR2 promoter regions. In contrast to murine TLR2, expression of human TLR2 in monocytes/macrophages is not induced by the proinflammatory stimuli LPS or macrophage-activating lipopeptide-2, and reporter activity of the promoter was not enhanced by stimuli-induced NF-kappaB activation in THP-1 or MonoMac-6 cells. Our findings provide an initial definition of the human TLR2 promoter and reveal profound differences in the regulation of an important pattern recognition molecule in humans and mice.
Subject(s)
Drosophila Proteins , Gene Expression Regulation , Macrophages/metabolism , Membrane Glycoproteins/genetics , Monocytes/metabolism , Receptors, Cell Surface/genetics , Transcription, Genetic , Alternative Splicing , Amino Acid Sequence , Base Sequence , CpG Islands , DNA Methylation , HeLa Cells , Humans , Molecular Sequence Data , NF-kappa B/metabolism , Promoter Regions, Genetic , RNA, Messenger/analysis , Toll-Like Receptor 2 , Toll-Like ReceptorsABSTRACT
The protein product of the CHI3L1 gene, human cartilage 39-kDa glycoprotein (HC-gp39), is a tissue-restricted, chitin-binding lectin and member of glycosyl hydrolase family 18. In contrast to many other monocyte/macrophage markers, its expression is absent in monocytes and strongly induced during late stages of human macrophage differentiation. To gain insights into the molecular mechanisms underlying its cell type-restricted and maturation-associated expression in macrophages, we initiated a detailed study of the proximal HC-gp39 promoter. Deletion analysis of reporter constructs in macrophage-like THP-1 cells localized a region directing high levels of macrophage-specific reporter gene expression to approximately 300 bp adjacent to the major transcriptional start site. The promoter sequence contained consensus binding sites for several known factors, and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1, and C/EBP proteins was detectable in gel shift assays. In vivo footprinting assays with dimethyl sulfate demonstrate that the protection of corresponding sequences was enhanced in macrophages compared with monocytes. Mutational analysis of transcription factor binding sites indicated a predominant role for a single Sp1 binding site in regulating HC-gp39 promoter activity. In addition, gel shift assays using nuclear extracts of monocytes and macrophages demonstrated that the binding of nuclear Sp1, but not Sp3, markedly increases during macrophage differentiation. Our results further highlight the important role of Sp1 in macrophage gene regulation.
Subject(s)
Cell Differentiation/genetics , Gene Expression Regulation , Glycoproteins/genetics , Macrophages/cytology , Adipokines , Animals , Base Sequence , Binding Sites , Cell Line , Cell Nucleus/chemistry , Chitinase-3-Like Protein 1 , Consensus Sequence , DNA/metabolism , DNA Footprinting , Drosophila/genetics , Electrophoretic Mobility Shift Assay , Gene Deletion , Gene Expression , Gene Expression Regulation/drug effects , Humans , Lectins , Molecular Sequence Data , Mutagenesis , Promoter Regions, Genetic/genetics , Sp1 Transcription Factor/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/metabolism , Transcription, Genetic , TransfectionABSTRACT
Toll-like receptor 3 (TLR3) belongs to a family of evolutionary conserved innate immune recognition molecules and recognizes double-stranded RNA, a molecular pattern associated with viral infections. Earlier studies suggested a differential expression pattern in men and mice; the molecular basis for this observation, however, was unknown. Here we demonstrate that species-specific differences in tissue expression and responses to lipopolysaccaride (LPS) coincide with the presence of different, evolutionary non-conserved promoter sequences in both species. Despite the overall unrelatedness of TLR3 promoter sequences, mRNA expression of both TLR3 orthologues was induced by interferons, particularly by interferon (IFN)-beta. The basal and IFN-beta-induced activation of promoters from both species largely depended on similar interferon regulatory factor (IRF) elements, which constitutively bound IRF-2 and recruited IRF-1 after stimulation. In murine macrophages, IFN-beta-induced TLR3 up-regulation required IFNAR1, STAT1, and in part IRF-1, but not the Janus kinase (Jak) family member Tyk2. We also show that LPS specifically up-regulates TLR3 expression in murine cells through the induction of autocrine/paracrine IFN-beta. In humans, however, IFN-beta-induced up-regulation of TLR3 was blocked by pretreatment with LPS, despite the efficient induction of IRF-1. Our findings reveal a mechanistic basis for the observed differences as well as similarities in TLR3 expression in men and mice. The IFN-beta-TLR3 link further suggests a role of TLR3 in innate and adaptive immune responses to viral infections. It will be interesting and important to clarify whether the observed differences in the transcriptional regulation of TLR3 influence innate immune responses in a species-specific manner.