ABSTRACT
The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.
Subject(s)
ADAM10 Protein , Animals , ADAM10 Protein/chemistry , ADAM10 Protein/metabolism , ADAM10 Protein/ultrastructure , Amyloid Precursor Protein Secretases/chemistry , Mammals/metabolism , Proteolysis , Tetraspanins/metabolism , HumansABSTRACT
Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.
Subject(s)
ADAM10 Protein/chemistry , Amyloid Precursor Protein Secretases/chemistry , Membrane Proteins/chemistry , Proteolysis , ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Crystallography, X-Ray , Humans , Membrane Proteins/metabolism , Models, Molecular , Receptors, Notch/metabolism , Signal TransductionABSTRACT
Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins or the molecular mechanisms underlying their various functions. Here, we report the crystal structure of human CD81, a full-length tetraspanin. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer membrane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.
Subject(s)
Cholesterol/metabolism , Molecular Dynamics Simulation , Tetraspanin 28/chemistry , Tetraspanin 28/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Models, ChemicalABSTRACT
Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9-12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.
Subject(s)
Drug Design , Protein Engineering , Proteins/chemical synthesis , Proteins/metabolism , 3T3 Cells , Animals , Cell Biology , Cell Survival , Computational Biology , Endocytosis , Escherichia coli/genetics , Escherichia coli/metabolism , In Vitro Techniques , Kinetics , Ligands , Mice , Microscopy, Atomic Force , Models, Molecular , Synthetic BiologyABSTRACT
Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition.
Subject(s)
Cell Cycle Proteins/chemistry , DNA-Binding Proteins/chemistry , DNA/chemistry , Nucleic Acid Conformation , Repressor Proteins/chemistry , Amino Acid Sequence/genetics , Base Sequence/genetics , Binding Sites/genetics , Cell Cycle Proteins/genetics , Crystallography, X-Ray , DNA/genetics , DNA-Binding Proteins/genetics , Forkhead Transcription Factors , Gene Expression Regulation/genetics , Humans , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Nucleotide Motifs/genetics , Regulatory Sequences, Nucleic Acid/genetics , Repressor Proteins/geneticsABSTRACT
Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.
Subject(s)
Cell Transformation, Neoplastic/genetics , Chromatin/genetics , Lymphoma, B-Cell/genetics , Oncogenes , Receptors, Notch/genetics , Triple Negative Breast Neoplasms/genetics , Binding Sites , Cell Lineage/genetics , Cell Proliferation/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Chromatin/metabolism , Chromatin Assembly and Disassembly , Cyclin D1/genetics , Cyclin D1/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , HEK293 Cells , Humans , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Mutation , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Notch/metabolism , Signal Transduction/genetics , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. DeltaMAX also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define DeltaMAX as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.
Subject(s)
Adaptor Proteins, Signal Transducing , Intercellular Signaling Peptides and Proteins , Humans , Animals , Mice , Ligands , Adaptor Proteins, Signal Transducing/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Calcium-Binding Proteins/metabolism , Signal Transduction/physiology , Receptors, Notch/metabolismABSTRACT
Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.
Subject(s)
Models, Molecular , Protein Structure, Tertiary , Receptors, Notch/chemistry , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Animals , Blotting, Western , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line, Tumor , Crystallography, X-Ray , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epitopes/chemistry , Epitopes/genetics , Epitopes/metabolism , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Ligands , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Secondary , Receptors, Notch/genetics , Receptors, Notch/metabolism , Sequence Homology, Amino Acid , Serrate-Jagged Proteins , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Wnt1 ProteinABSTRACT
Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.
Subject(s)
Receptors, Notch/metabolism , Small Molecule Libraries/pharmacology , Transcriptional Activation/drug effects , Animals , Apoptosis/drug effects , Binding Sites , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drosophila , Drug Resistance, Neoplasm/drug effects , HeLa Cells , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/chemistry , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Intestine, Small/drug effects , Intestine, Small/metabolism , Mice , Mutation , Phenotype , Protein Multimerization , Signal Transduction/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic useABSTRACT
Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Amidohydrolases/metabolism , Gastrointestinal Microbiome/physiology , Amidohydrolases/physiology , Animals , Bacteria/enzymology , Bile Acids and Salts/metabolism , Drug Design , Female , Humans , Male , Mice , Mice, Inbred C57BL , Small Molecule LibrariesABSTRACT
SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.
Subject(s)
Antineoplastic Agents/pharmacology , Methanol/analogs & derivatives , Neoplasms/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrazines/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Molecular Targeted Therapy , Mutation , Neoplasms/metabolism , Neoplasms/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Proteolysis , Signal TransductionABSTRACT
Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads not be restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow-extension strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allow assignment of tethered substrates to singly captured and multiply tethered bins, with the fraction of fully mobile, single-tether substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiple-tether beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.
Subject(s)
ADAM17 Protein/chemistry , Endopeptidases/chemistry , Peptides/analysis , Proteolysis , Single Molecule Imaging/methods , DNA/chemistry , Humans , Magnetic Phenomena , Microfluidics/methods , Motion , Peptides/chemistry , Potyvirus/enzymology , Proof of Concept StudyABSTRACT
Research in the last several years has shown that Notch proteolysis, and thus Notch activation, is conformationally controlled by the extracellular juxtamembrane NRR of Notch, which sterically occludes the S2 protease site until ligand binds. The question of how conformational exposure of the protease site is achieved during physiologic activation, and thus how normal activation is bypassed in disease pathogenesis, has been the subject of intense study in the last several years, and is the subject of this chapter. Here, we summarize the structural features of the NRR domains of Notch receptors that establish the autoinhibited state and then review a number of recent studies aimed at testing the mechanotransduction model for Notch signaling using force spectroscopy and molecular tension sensors.
Subject(s)
Proteolysis , Receptors, Notch , Signal Transduction/physiology , Animals , Humans , Protein Domains , Receptors, Notch/chemistry , Receptors, Notch/genetics , Receptors, Notch/metabolism , Structure-Activity RelationshipABSTRACT
Notch signaling regulates myriad cellular functions by activating transcription, yet how Notch selectively activates different transcriptional targets is poorly understood. The core Notch transcriptional activation complex can bind DNA as a monomer, but it can also dimerize on DNA-binding sites that are properly oriented and spaced. However, the significance of Notch dimerization is unknown. Here, we show that dimeric Notch transcriptional complexes are required for T-cell maturation and leukemic transformation but are dispensable for T-cell fate specification from a multipotential precursor. The varying requirements for Notch dimerization result from the differential sensitivity of specific Notch target genes. In particular, c-Myc and pre-T-cell antigen receptor α (Ptcra) are dimerization-dependent targets, whereas Hey1 and CD25 are not. These findings identify functionally important differences in the responsiveness among Notch target genes attributable to the formation of higher-order complexes. Consequently, it may be possible to develop a new class of Notch inhibitors that selectively block outcomes that depend on Notch dimerization (e.g., leukemogenesis).
Subject(s)
Protein Multimerization , Receptor, Notch1/chemistry , Receptor, Notch1/metabolism , T-Lymphocytes/metabolism , Animals , Base Sequence , Binding Sites , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Flow Cytometry , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Models, Molecular , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptor, Notch1/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Signal Transduction/genetics , Signal Transduction/physiology , T-Lymphocytes/cytology , Transcription, GeneticABSTRACT
Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.
Subject(s)
Fucosyltransferases/chemistry , Fucosyltransferases/genetics , Hyperpigmentation/genetics , Mutation , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction/genetics , Skin Diseases, Genetic/genetics , Skin Diseases, Papulosquamous/genetics , Carcinogenesis/genetics , Carcinogenesis/metabolism , Fucosyltransferases/deficiency , Fucosyltransferases/metabolism , Humans , Hyperpigmentation/metabolism , Ligands , Protein Conformation , Skin Diseases, Genetic/metabolism , Skin Diseases, Papulosquamous/metabolismABSTRACT
The PTPN11 oncogene encodes the cytoplasmic protein tyrosine phosphatase SHP2, which, through its role in multiple signaling pathways, promotes the progression of hematological malignancies and other cancers. Here, we employ high-throughput screening to discover a lead chemical scaffold, the benzothiazolopyrimidones, that allosterically inhibits this oncogenic phosphatase by simultaneously engaging the C-SH2 and PTP domains. We improved our lead to generate an analogue that better suppresses SHP2 activity in vitro. Suppression of Erk phopsphorylation by the lead compound is also consistent with SHP2 inhibition in AML cells. Our findings provide an alternative starting point for therapeutic intervention and will catalyze investigations into the relationship between SHP2 conformational regulation, activity, and disease progression.
Subject(s)
Benzothiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrimidinones/pharmacology , Allosteric Regulation/drug effects , Benzothiazoles/chemical synthesis , Benzothiazoles/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Structure-Activity RelationshipABSTRACT
The main oncogenic driver in T-lymphoblastic leukemia is NOTCH1, which activates genes by forming chromatin-associated Notch transcription complexes. Gamma-secretase-inhibitor treatment prevents NOTCH1 nuclear localization, but most genes with NOTCH1-binding sites are insensitive to gamma-secretase inhibitors. Here, we demonstrate that fewer than 10% of NOTCH1-binding sites show dynamic changes in NOTCH1 occupancy when T-lymphoblastic leukemia cells are toggled between the Notch-on and -off states with gamma-secretase inhibiters. Dynamic NOTCH1 sites are functional, being highly associated with Notch target genes, are located mainly in distal enhancers, and frequently overlap with RUNX1 binding. In line with the latter association, we show that expression of IL7R, a gene with key roles in normal T-cell development and in T-lymphoblastic leukemia, is coordinately regulated by Runx factors and dynamic NOTCH1 binding to distal enhancers. Like IL7R, most Notch target genes and associated dynamic NOTCH1-binding sites cooccupy chromatin domains defined by constitutive binding of CCCTC binding factor, which appears to restrict the regulatory potential of dynamic NOTCH1 sites. More remarkably, the majority of dynamic NOTCH1 sites lie in superenhancers, distal elements with exceptionally broad and high levels of H3K27ac. Changes in Notch occupancy produces dynamic alterations in H3K27ac levels across the entire breadth of superenhancers and in the promoters of Notch target genes. These findings link regulation of superenhancer function to NOTCH1, a master regulatory factor and potent oncoprotein in the context of immature T cells, and delineate a generally applicable roadmap for identifying functional Notch sites in cellular genomes.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/physiology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Multiprotein Complexes/metabolism , Receptor, Notch1/metabolism , Signal Transduction/physiology , Binding Sites/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Primers/genetics , Gene Expression Regulation/genetics , Humans , Luciferases , Plasmids/genetics , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Receptors, Interleukin-7/metabolism , Signal Transduction/geneticsABSTRACT
Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Leukemic/genetics , Genes, myc , Neoplasm Proteins/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptor, Notch1/metabolism , Animals , Base Sequence , Cell Cycle Proteins , Cell Line, Tumor , Chromatin Immunoprecipitation , Genes, Reporter , Genome-Wide Association Study , Histones/metabolism , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Models, Molecular , Molecular Sequence Data , Neoplasm Proteins/metabolism , Nuclear Proteins/antagonists & inhibitors , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Promoter Regions, Genetic/genetics , Protein Conformation , Receptor, Notch1/antagonists & inhibitors , Sequence Alignment , Sequence Homology, Nucleic Acid , Transcription Factors/antagonists & inhibitors , Transcription, GeneticABSTRACT
Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.