A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.

Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches.

The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.

Variability in Streptavidin-Sepharose Matrix Quality Can Significantly Affect Proximity-Dependent Biotinylation (BioID) Data.

Due to their ease of use and high binding affinity, streptavidin-based purification tools have become widely used for isolating biotinylated compounds from complex mixtures. We and others routinely use streptavidin-sepharose matrices to isolate biotinylated polypeptides generated in proximity-dependent biotinylation approaches, such as BioID or APEX. However, we noted sporadic, substantial variation in the quality of BioID experiments performed in the same laboratories over time, using seemingly identical protocols. Identifying the source of this problem, here, we highlight considerable variability in streptavidin contamination derived from different production lots of streptavidin-sepharose beads from the same manufacturer and demonstrate that high levels of streptavidin peptide contamination can have detrimental effects on BioID data. We also describe two simple, rapid approaches to assess the degree of streptavidin "shedding" from individual lots of the sepharose matrix before use to avoid the use of lower quality reagent.

A Versatile Lentiviral Delivery Toolkit for Proximity-dependent Biotinylation in Diverse Cell Types.

Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents.

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin.

The Crumbs complex is an important determinant of epithelial apical-basal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADP-ribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane.

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.

Transcription factor-mediated epigenetic reprogramming.

Input from various signaling pathways in conjunction with specific transcription factors (TFs), noncoding RNAs, and epigenetic modifiers governs the maintenance of cellular identity. Endogenous or exogenous TFs operate within certain boundaries, which are set, in part, by the cell type-specific epigenetic landscape. Ectopic expression of selected TFs can override the cellular identity and induce reprogramming to alternative fates. In this minireview, we summarize many of the classic examples and a large number of recent studies that have taken advantage of TF-mediated reprogramming to produce cell types of biomedical relevance.

Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.

A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions.

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.

A simple protein-based surrogate neutralization assay for SARS-CoV-2.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

Microtubules regulate PI-3K activity and recruitment to the phagocytic cup during Fcgamma receptor-mediated phagocytosis in nonelicited macrophages.

Phagocytosis is a complex sequence of events involving coordinated remodeling of the plasma membrane with the underlying cytoskeleton. Although the role of the actin cytoskeleton is becoming increasingly elucidated, the role of microtubules (MTs) remains poorly understood. Here, we examine the role of MTs during FcgammaR-mediated phagocytosis in RAW264.7 mouse macrophages. We observe that MTs extend into the phagosomal cups. The MT-depolymerizing agents, colchicine and nocodazole, cause a sizeable reduction in phagocytosis of large particles in RAW264.7 cells. Phagocytosis in primed macrophages is unaffected by MT-depolymerizing agents. However, activation of macrophages coincides with an increased population of drug-stable MTs, which persist in functional phagocytic cups. Scanning electron microscopy analysis of unprimed macrophages reveals that pseudopod formation is reduced markedly following colchicine treatment, which is not a consequence of cell rounding. MT depolymerization in these cells does not affect particle binding, Syk, or Grb2-associated binder 2 recruitment or phosphotyrosine accumulation at the site of phagocytosis. Ras activation also proceeds normally in macrophages treated with colchicine. However, MT disruption causes a decrease in accumulation of AKT-pleckstrin homology-green fluorescent protein, a probe that binds to PI-3K products at the sites of particle binding. A corresponding decline in activated AKT is observed in colchicine-treated cells using immunoblotting with a phospho-specific-AKT (ser473) antibody. Furthermore, the translocation of the p85alpha regulatory subunit of PI-3K is reduced at the phagocytic cup in colchicine-treated cells. These findings suggest that MTs regulate the recruitment and localized activity of PI-3K during pseudopod formation.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry.

Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.

Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.

BACKGROUND: Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution. RESULTS: Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop. CONCLUSIONS: TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs.

Distinct polarity cues direct Taz/Yap and TGFß receptor localization to differentially control TGFß-induced Smad signaling.

We and others have shown that the Hippo pathway effectors TAZ and YAP direct Smad activity to regulate TGFß family-induced cellular responses in stem cell and cancer biology. In polarized epithelial cells we showed that the Crumbs complex promotes Hippo-dependent cytoplasmic TAZ/YAP localization that restricts TGFß-induced Smad nuclear accumulation and activity. In this Developmental Cell issue, basal-lateral restriction of TGFß receptors is proposed as the sole mechanism suppressing Smad signaling in epithelial cells. Here we show that basal recruitment of TGFß receptors occurs subsequent to Hippo-dependent suppression of Smad activity by cytoplasmic TAZ/YAP. Our results demonstrate that receptor sequestration and Hippo control of activated Smads are distinct events regulating TGFß signaling in polarized epithelia and raise interesting questions about the function of these pathways in controlling Smad signaling in development, homeostasis, and disease. This Matters Arising Response addresses the Nallet-Staub et al. (2015) Matters Arising, published concurrently in Developmental Cell.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome.

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome. Here we demonstrate a loss of C4S, as well as a reduction in C4ST-1 mRNA and protein expression, in primary fibroblasts from Costello syndrome patients. We go on to show that expression of oncogenic HRAS in normal fibroblasts can repress C4ST-1 expression, whereas interference with oncogenic HRAS signaling in Costello syndrome fibroblasts elevated C4ST-1 expression, thus identifying C4ST-1 as a negatively regulated target gene of HRAS signaling. Importantly, we show that forced expression of C4ST-1 in Costello fibroblasts could rescue the proliferation and elastogenesis defects associated with oncogenic HRAS signaling in these cells. Our results indicate reduced C4ST-1 expression and chondroitin sulfation imbalance mediating the effects of oncogenic HRAS signaling in the pathogenesis of Costello syndrome. Thus, our work identifies C4ST-1-dependent chondroitin sulfation as a downstream vulnerability in oncogenic RAS signaling, which might be pharmacologically exploited in future treatments of not only Costello syndrome and other RASopathies, but also human cancers associated with activating RAS mutations.