Systematic identification of factors for provirus silencing in embryonic stem cells.

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.

Functional genomics identifies specific vulnerabilities in PTEN-deficient breast cancer.

BACKGROUND: Phosphatase and tensin homolog (PTEN) is one of the most frequently inactivated tumor suppressors in breast cancer. While PTEN itself is not considered a druggable target, PTEN synthetic-sick or synthetic-lethal (PTEN-SSL) genes are potential drug targets in PTEN-deficient breast cancers. Therefore, with the aim of identifying potential targets for precision breast cancer therapy, we sought to discover PTEN-SSL genes present in a broad spectrum of breast cancers. METHODS: To discover broad-spectrum PTEN-SSL genes in breast cancer, we used a multi-step approach that started with (1) a genome-wide short interfering RNA (siRNA) screen of ~ 21,000 genes in a pair of isogenic human mammary epithelial cell lines, followed by (2) a short hairpin RNA (shRNA) screen of ~ 1200 genes focused on hits from the first screen in a panel of 11 breast cancer cell lines; we then determined reproducibility of hits by (3) identification of overlaps between our results and reanalyzed data from 3 independent gene-essentiality screens, and finally, for selected candidate PTEN-SSL genes we (4) confirmed PTEN-SSL activity using either drug sensitivity experiments in a panel of 19 cell lines or mutual exclusivity analysis of publicly available pan-cancer somatic mutation data. RESULTS: The screens (steps 1 and 2) and the reproducibility analysis (step 3) identified six candidate broad-spectrum PTEN-SSL genes (PIK3CB, ADAMTS20, AP1M2, HMMR, STK11, and NUAK1). PIK3CB was previously identified as PTEN-SSL, while the other five genes represent novel PTEN-SSL candidates. Confirmation studies (step 4) provided additional evidence that NUAK1 and STK11 have PTEN-SSL patterns of activity. Consistent with PTEN-SSL status, inhibition of the NUAK1 protein kinase by the small molecule drug HTH-01-015 selectively impaired viability in multiple PTEN-deficient breast cancer cell lines, while mutations affecting STK11 and PTEN were largely mutually exclusive across large pan-cancer data sets. CONCLUSIONS: Six genes showed PTEN-SSL patterns of activity in a large proportion of PTEN-deficient breast cancer cell lines and are potential specific vulnerabilities in PTEN-deficient breast cancer. Furthermore, the NUAK1 PTEN-SSL vulnerability identified by RNA interference techniques can be recapitulated and exploited using the small molecule kinase inhibitor HTH-01-015. Thus, NUAK1 inhibition may be an effective strategy for precision treatment of PTEN-deficient breast tumors.

Short O-GalNAc glycans: regulation and role in tumor development and clinical perspectives.

BACKGROUND: While the underlying causes of cancer are genetic modifications, changes in cellular states mediate cancer development. Tumor cells display markedly changed glycosylation states, of which the O-GalNAc glycans called the Tn and TF antigens are particularly common. How these antigens get over-expressed is not clear. The expression levels of glycosylation enzymes fail to explain it. SCOPE OF REVIEW: We describe the regulation of O-GalNAc glycosylation initiation and extension with emphasis on the initiating enzymes ppGalNAcTs (GALNTs), and introduce the GALA pathway--a change in GALNTs compartmentation within the secretory pathway that regulates Tn levels. We discuss the roles of O-GalNAc glycans and GALNTs in tumorigenic processes and finally consider diagnostic and therapeutic perspectives. MAJOR CONCLUSIONS: Contrary to a common hypothesis, short O-glycans in tumors are not the result of an incomplete glycosylation process but rather reveal the activation of regulatory pathways. Surprisingly, high Tn levels reveal a major shift in the O-glycoproteome rather than a shortening of O-glycans. These changes are driven by membrane trafficking events. GENERAL SIGNIFICANCE: Many attempts to use O-glycans for biomarker, antibody and therapeutic vaccine development have been made, but suffer limitations including poor sensitivity and/or specificity that may in part derive from lack of a mechanistic understanding. Deciphering how short O-GalNAc glycans are regulated would open new perspectives to exploit this biology for therapeutic usage. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

ScreenSifter: analysis and visualization of RNAi screening data.

BACKGROUND: RNAi screening is a powerful method to study the genetics of intracellular processes in metazoans. Technically, the approach has been largely inspired by techniques and tools developed for compound screening, including those for data analysis. However, by contrast with compounds, RNAi inducing agents can be linked to a large body of gene-centric, publically available data. However, the currently available software applications to analyze RNAi screen data usually lack the ability to visualize associated gene information in an interactive fashion. RESULTS: Here, we present ScreenSifter, an open-source desktop application developed to facilitate storing, statistical analysis and rapid and intuitive biological data mining of RNAi screening datasets. The interface facilitates meta-data acquisition and long-term safe-storage, while the graphical user interface helps the definition of a hit list and the visualization of biological modules among the hits, through Gene Ontology and protein-protein interaction analyses. The application also allows the visualization of screen-to-screen comparisons. CONCLUSIONS: Our software package, ScreenSifter, can accelerate and facilitate screen data analysis and enable discovery by providing unique biological data visualization capabilities.

RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.

The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication.

Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.

RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming.

Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.

RNAi screens for genes involved in Golgi glycosylation.

RNAi screening has gained popularity in recent years, due to its usefulness in systematic investigations of biological pathways. Combined with high-content screening and advances in imaging and analysis methods, it can enable detailed genetic characterization of cellular processes such as protein glycosylation, a major function of the Golgi apparatus. Glycosylation concerns about one third of all human proteins and regulates various cellular behaviors. Yet the methods available to study it are limited and not easily accessible. In this chapter, we detail a step-by-step method to systematically and quantitatively investigate glycosylation using fluorescent lectin staining, following high-throughput RNAi-based downregulation of gene activities. We also provide a workflow for downstream analysis of the data generated.

The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

Presynaptic regulation of quantal size: K+/H+ exchange stimulates vesicular glutamate transport.

The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δµ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δµ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δµ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.

Vesicular glutamate transport promotes dopamine storage and glutamate corelease in vivo.

Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl(-), indicating a distinct, presynaptic mechanism to regulate quantal size.

Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate.

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.