ABSTRACT
Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the D4Z4 locus leading to aberrant double homeobox 4 (DUX4) expression in skeletal muscle. Activation of this early embryonic transcription factor results in the expression of its target genes causing muscle fiber death. Although progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent, and specific inhibitors of p38α/ß, we show a robust reduction of DUX4 expression, activity, and cell death across patient-derived FSHD1 and FSHD2 lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/ß inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/ß inhibitors as effective therapeutics to treat FSHD at its root cause. SIGNIFICANCE STATEMENT: Using patient-derived facioscapulohumeral muscular dystrophy (FSHD) myotubes, we characterize the pharmacological relationships between p38α/ß inhibition, double homeobox 4 (DUX4) expression, its downstream transcriptional program, and muscle cell death. p38α/ß inhibition results in potent and specific DUX4 downregulation across multiple genotypes without significant effects in the process of myogenesis in vitro. These findings highlight the potential of p38α/ß inhibitors for the treatment of FSHD, a condition that today has no approved therapies.
Subject(s)
Homeodomain Proteins/metabolism , Muscular Dystrophy, Facioscapulohumeral/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Death/physiology , Cell Line , Gene Expression Regulation/physiology , Humans , Muscle Cells/metabolism , Muscle, Skeletal/metabolismABSTRACT
Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.
Subject(s)
Cell-Penetrating Peptides/pharmacology , Peptide Fragments/pharmacology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Animals , Basic-Leucine Zipper Transcription Factors/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell-Penetrating Peptides/pharmacokinetics , Cell-Penetrating Peptides/therapeutic use , DNA/metabolism , Disease Models, Animal , E-Box Elements/genetics , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice, Inbred C57BL , Peptide Fragments/administration & dosage , Peptide Fragments/pharmacokinetics , Peptide Fragments/therapeutic use , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Multimerization/drug effects , Proto-Oncogene Proteins c-myc/administration & dosage , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/pharmacokinetics , Proto-Oncogene Proteins c-myc/pharmacology , Proto-Oncogene Proteins c-myc/therapeutic useABSTRACT
Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of Pparg and Cebpa, thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the Pparg locus by CRISPRi demonstrates a critical role for these enhancers in the control of Pparg gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition.
Subject(s)
Adipocytes/physiology , Adipose Tissue/cytology , Gene Expression Regulation/physiology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Adipogenesis , Adipose Tissue/physiology , Animals , Cell Differentiation , Male , MiceABSTRACT
Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.
Subject(s)
Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Phenylenediamines/pharmacology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Pyrimidines/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Core Binding Factor Alpha 2 Subunit/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cysteine/metabolism , Humans , Jurkat Cells , Phosphorylation/drug effectsABSTRACT
We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.
Subject(s)
Benzeneacetamides/pharmacology , Benzhydryl Compounds/pharmacology , Digoxin/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Gene Regulatory Networks/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Multiple Sclerosis/drug therapy , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , T-Lymphocyte Subsets/drug effects , Th17 Cells/drug effects , Androstenols/chemistry , Animals , Benzeneacetamides/chemistry , Benzhydryl Compounds/chemistry , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Cytokines/metabolism , Digoxin/chemistry , Encephalomyelitis, Autoimmune, Experimental/immunology , Heterocyclic Compounds, 4 or More Rings/chemistry , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiple Sclerosis/immunology , Myelin-Oligodendrocyte Glycoprotein/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Peptide Fragments/immunology , Protein Binding/drug effects , Structure-Activity Relationship , Systems Biology , T-Lymphocyte Subsets/immunology , Th17 Cells/immunology , Transcription, Genetic/drug effects , Transcriptional Activation/drug effectsABSTRACT
Most transcription factors specify the subset of genes that will be actively transcribed in the cell by stimulating transcription initiation at these genes, but MYC has a fundamentally different role. MYC binds E-box sites in the promoters of active genes and stimulates recruitment of the elongation factor P-TEFb and thus transcription elongation. Consequently, rather than specifying the set of genes that will be transcribed in any particular cell, MYC's predominant role is to increase the production of transcripts from active genes. This increase in the transcriptional output of the cell's existing gene expression program, called transcriptional amplification, has a profound effect on proliferation and other behaviors of a broad range of cells. Transcriptional amplification may reduce rate-limiting constraints for tumor cell proliferation and explain MYC's broad oncogenic activity among diverse tissues.
Subject(s)
Genes, myc/physiology , Proto-Oncogene Proteins c-myc/metabolism , Transcription Elongation, Genetic , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Gene Amplification , Gene Expression Regulation, Neoplastic , Humans , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/metabolismABSTRACT
A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.
Subject(s)
Chromatin/genetics , DNA/genetics , Proteins/genetics , Transcription Factors/genetics , Binding Sites/genetics , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Ligands , Protein Binding/geneticsABSTRACT
Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of bromodomain and extra-terminal domain (BET) proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of bromodomain 4 (BRD4) at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease.
Subject(s)
Enhancer Elements, Genetic , Lymphoma, Large B-Cell, Diffuse/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Azepines/pharmacology , Cell Cycle Proteins , DNA-Binding Proteins/genetics , Genes, myc , Humans , Promoter Regions, Genetic , Proto-Oncogene Proteins c-bcl-6 , Trans-Activators/physiology , Transcription, Genetic , Triazoles/pharmacologyABSTRACT
MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.
Subject(s)
Fibroblasts/cytology , Gene Expression Regulation , MicroRNAs/genetics , MicroRNAs/metabolism , Nuclear Receptor Subfamily 6, Group A, Member 1/genetics , Nuclear Receptor Subfamily 6, Group A, Member 1/metabolism , Animals , Antigens, Neoplasm/metabolism , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Fibroblasts/metabolism , Gene Deletion , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Protein Binding , Ribonuclease III/genetics , Ribonuclease III/metabolismABSTRACT
Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity.
Subject(s)
Cell Lineage , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic , Mediator Complex/metabolism , Transcription Factors/metabolism , Animals , B-Lymphocytes/metabolism , Cell Line , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Mice , Transcription, GeneticABSTRACT
Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.
Subject(s)
Gene Expression Profiling/methods , Genome-Wide Association Study , Humans , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myc/genetics , Sequence Analysis, RNA , Transcription, GeneticABSTRACT
Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Proto-Oncogene Proteins c-myc/metabolism , Cell Line, Tumor , Cell Proliferation , Enhancer Elements, Genetic , Humans , Neoplasms/pathology , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.
Subject(s)
RNA Polymerase II/physiology , HeLa Cells , Humans , RNA Polymerase II/metabolism , Transcription Factors, TFII/metabolism , Transcription, GeneticABSTRACT
Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation/immunology , Self Tolerance/immunology , T-Lymphocytes/immunology , Thymus Gland/cytology , Transcription Factors/metabolism , Animals , Cell Line , Chromatin Immunoprecipitation , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Kruppel-Like Factor 4 , Mice , Mice, Inbred C57BL , Microarray Analysis , T-Lymphocytes/cytology , AIRE ProteinABSTRACT
MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Multiple Myeloma/drug therapy , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Azepines/chemistry , Azepines/pharmacology , Benzodiazepines/chemistry , Benzodiazepines/pharmacology , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Transcriptional Activation/drug effects , Triazoles/chemistry , Triazoles/pharmacologyABSTRACT
Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.
Subject(s)
Melanoma/genetics , Melanoma/pathology , Neural Crest/enzymology , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Transcription, Genetic , Amino Acid Substitution , Animals , Animals, Genetically Modified , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , Dihydroorotate Dehydrogenase , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Genes, p53/genetics , Humans , Isoxazoles/pharmacology , Isoxazoles/therapeutic use , Leflunomide , Melanoma/drug therapy , Melanoma/enzymology , Mice , Neural Crest/drug effects , Neural Crest/metabolism , Neural Crest/pathology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/pathology , Transcription, Genetic/drug effects , Transcription, Genetic/physiology , Xenograft Model Antitumor Assays , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Embryonic Stem Cells/metabolism , Gene Expression Regulation/genetics , Mediator Complex/metabolism , Animals , Cell Cycle Proteins/genetics , Cells, Cultured , Chromatin/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA/chemistry , DNA/genetics , DNA/metabolism , Embryonic Stem Cells/cytology , Enhancer Elements, Genetic/genetics , Fibroblasts , Mediator Complex/genetics , Mice , Nucleic Acid Conformation , Organ Specificity , Promoter Regions, Genetic/genetics , Protein Binding , CohesinsABSTRACT
Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.
Subject(s)
Proto-Oncogene Proteins c-myc/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic , Animals , Cell Proliferation , Chromatin Immunoprecipitation , Embryonic Stem Cells/metabolism , Humans , Mice , Nuclear Proteins/metabolism , Octamer Transcription Factor-3/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcriptional Elongation FactorsABSTRACT
Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-Seq) and functional analysis revealed that SetDB1 and histone H3K9-methylated nucleosomes occupy and repress genes encoding developmental regulators. These SetDB1-occupied genes are a subset of the "bivalent" genes, which contain nucleosomes with H3K4me3 (H3K4 trimethylation) and H3K27me3 modifications catalyzed by Trithorax and Polycomb group proteins, respectively. These genes are subjected to repression by both Polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state.
Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/enzymology , Gene Expression Regulation, Developmental , Methyltransferases/metabolism , Protein Methyltransferases/metabolism , Animals , Chromatin/metabolism , Chromatin Immunoprecipitation , Histone-Lysine N-Methyltransferase , Mice , Nucleosomes/enzymologyABSTRACT
Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site-associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.