Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation.

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

Primary cilia on muscle stem cells are critical to maintain regenerative capacity and are lost during aging.

During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We found that the ability of MuSCs to regenerate is regulated by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia inhibited MuSC proliferation in vitro and severely impaired injury-induced muscle regeneration in vivo. In aged muscle, a cell intrinsic defect in MuSC ciliation was associated with the decrease in regenerative capacity. Exogenous activation of Hedgehog signaling, known to be localized in the primary cilium, promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG1.3) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aged MuSCs and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.

Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing.

Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.

Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains.

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

Combined Proteomic and Genetic Interaction Mapping Reveals New RAS Effector Pathways and Susceptibilities.

Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.This article is highlighted in the In This Issue feature, p. 1775.

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release.

Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5ß1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5ß1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.

E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.

The CEP19-RABL2 GTPase Complex Binds IFT-B to Initiate Intraflagellar Transport at the Ciliary Base.

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT.

Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis.

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.

Comparative Proteomics Reveals Strain-Specific ß-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex.

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and ß-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated ß-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets ß-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote ß-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.

Fibronectin matrix mimetics promote full-thickness wound repair in diabetic mice.

During tissue repair, fibronectin is converted from a soluble, inactive form into biologically active extracellular matrix (ECM) fibrils through a cell-dependent process. ECM fibronectin promotes numerous cell processes that are critical to tissue repair and regulates the assembly of other proteins into the matrix. Nonhealing wounds show reduced levels of ECM fibronectin. To functionally mimic ECM fibronectin, a series of fibronectin matrix mimetics was developed by directly coupling the matricryptic, heparin-binding fragment of the first type III repeat of fibronectin (FNIII1H) to various sequences from the integrin-binding domain (FNIII8-10). The recombinant proteins were produced as glutathione-S-transferase (GST)-tagged fusion proteins for ease of production and purification. Full-thickness, excisional wounds were produced in genetically diabetic mice, and fibronectin matrix mimetics were applied directly to the wounds. A significant enhancement of wound closure was observed by day 9 in response to GST/III1H,8-10 versus GST-treated controls (73.9%±4.1% vs. 58.1%±4.7% closure, respectively). Two weeks after injury, fibronectin matrix mimetic-treated wounds had developed a multi-layered epithelium that completely covered the wound space. Furthermore, significant increases in granulation tissue thickness were observed in response to treatment with GST/III1H,8-10 (4.05±0.93-fold), GST/III1H,8,10 (2.91±0.49-fold), or GST/III1H,8(RGD) (3.55±0.59-fold) compared with GST controls, and was accompanied by dense collagen deposition, the presence of myofibroblasts, and functional vasculature. Thus, the recombinant fibronectin matrix analogs normalized the impairment in repair observed in this chronic wound model and may provide a new approach to accelerate the healing of diabetic wounds.