Temporal dynamics and stoichiometry in human Notch signaling from Notch synaptic complex formation to nuclear entry of the Notch intracellular domain.

Mammalian Notch signaling occurs when the binding of Delta or Jagged to Notch stimulates the proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to control target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20 min after contact. Synapse formation preceded the entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after ∼45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with spatiotemporal precision.

Temporal Dynamics and Stoichiometry in Notch Signaling - from Notch Synaptic Complex Formation to NICD Nuclear Entry.

Mammalian Notch signaling occurs when binding of Delta or Jagged to Notch stimulates proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to regulate target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20 min after contact. Synapse formation preceded entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after ∼45 min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with unprecedented spatiotemporal precision.

A spatiotemporal Notch interaction map from plasma membrane to nucleus.

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity.

Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

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.

Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling.

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.

MAML1-Dependent Notch-Responsive Genes Exhibit Differing Cofactor Requirements for Transcriptional Activation.

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Knockout of Mastermind-like 1 (MAML1) eliminates Notch-responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of MAML1 variants in knockout cells identifies residues 151 to 350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the histone acetyltransferase (HAT) domain of p300 rescues expression of HES4 but not DTX1, suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1 region from residues 151 to 350 for Notch-dependent transcriptional induction and reveal differential requirements for MAML1-dependent recruitment activities at different Notch-responsive loci, highlighting the molecular complexity of Notch-stimulated transcription.

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations.

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.

Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex.

RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing.

Biogenesis of telomerase ribonucleoproteins.

Telomerase adds simple-sequence repeats to the ends of linear chromosomes to counteract the loss of end sequence inherent in conventional DNA replication. Catalytic activity for repeat synthesis results from the cooperation of the telomerase reverse transcriptase protein (TERT) and the template-containing telomerase RNA (TER). TERs vary widely in sequence and structure but share a set of motifs required for TERT binding and catalytic activity. Species-specific TER motifs play essential roles in RNP biogenesis, stability, trafficking, and regulation. Remarkably, the biogenesis pathways that generate mature TER differ across eukaryotes. Furthermore, the cellular processes that direct the assembly of a biologically functional telomerase holoenzyme and its engagement with telomeres are evolutionarily varied and regulated. This review highlights the diversity of strategies for telomerase RNP biogenesis, RNP assembly, and telomere recruitment among ciliates, yeasts, and vertebrates and suggests common themes in these pathways and their regulation.

An enhanced H/ACA RNP assembly mechanism for human telomerase RNA.

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.

Specificity and stoichiometry of subunit interactions in the human telomerase holoenzyme assembled in vivo.

The H/ACA motif of human telomerase RNA (hTR) directs specific pathways of endogenous telomerase holoenzyme assembly, function, and regulation. Similarities between hTR and other H/ACA RNAs have been established, but differences have not been explored even though unique features of hTR H/ACA RNP assembly give rise to telomerase deficiency in human disease. Here, we define hTR H/ACA RNA and RNP architecture using RNA accumulation, RNP affinity purification, and primer extension activity assays. First, we evaluate alternative folding models for the hTR H/ACA motif 5' hairpin. Second, we demonstrate an unanticipated and surprisingly general asymmetry of 5' and 3' hairpin requirements for H/ACA RNA accumulation. Third, we establish that hTR assembles not one but two sets of all four of the H/ACA RNP core proteins, dyskerin, NOP10, NHP2, and GAR1. Fourth, we address a difference in predicted specificities of hTR association with the holoenzyme subunit WDR79/TCAB1. Together, these results complete the analysis of hTR elements required for active RNP biogenesis and define the interaction specificities and stoichiometries of all functionally essential human telomerase holoenzyme subunits. This study uncovers unexpected similarities but also differences between telomerase and other H/ACA RNPs that allow a unique specificity of telomerase biogenesis and regulation.