Publications by authors named "Martin Kampmann"

62 Publications

Functional genomics screen identifies proteostasis targets that modulate prion protein (PrP) stability.

Cell Stress Chaperones 2021 Mar 5;26(2):443-452. Epub 2021 Feb 5.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, 94158, USA.

Prion protein (PrP) adopts either a helical conformation (PrP) or an alternative, beta sheet-rich, misfolded conformation (PrP). The PrP form has the ability to "infect" PrP and force it into the misfolded state. Accumulation of PrP is associated with a number of lethal neurodegenerative disorders, including Creutzfeldt-Jacob disease (CJD). Knockout of PrP protects cells and animals from PrP infection; thus, there is interest in identifying factors that regulate PrP stability, with the therapeutic goal of reducing PrP levels and limiting infection by PrP. Here, we assembled a short-hairpin RNA (shRNA) library composed of 25+ shRNA sequences for each of 133 protein homeostasis (aka proteostasis) factors, such as molecular chaperones and co-chaperones. This Proteostasis shRNA Library was used to identify regulators of PrP stability in HEK293 Hu129M cells. Strikingly, the screen identified a number of Hsp70 family members and their co-chaperones as putative targets. Indeed, a chemical pan-inhibitor of Hsp70s reduced PrP levels and limited conversion to PrP in N2a cells. These results implicate specific proteostasis sub-networks, especially the Hsp70 system, as potential new targets for the treatment of CJD. More broadly, the Proteostasis shRNA Library might be a useful tool for asking which proteostasis factors are important for a given protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12192-021-01191-8DOI Listing
March 2021

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted an unbiased CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is an essential node in the cellular response to SARS-CoV-2 infection. BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells. BRD2 also controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates ACE2 levels. It is possible that the previously reported interaction between the viral E protein and BRD2 evolved to manipulate the transcriptional host response during SARS-CoV-2 infection. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.01.19.427194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836110PMC
January 2021

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes.

J Cell Biol 2021 Feb;220(2)

Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202006180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816647PMC
February 2021

Molecular characterization of selectively vulnerable neurons in Alzheimer's disease.

Nat Neurosci 2021 Feb 11;24(2):276-287. Epub 2021 Jan 11.

Institute for Neurodegenerative Disease, University of California, San Francisco, San Francisco, CA, USA.

Alzheimer's disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus-brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively-from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41593-020-00764-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854528PMC
February 2021

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling.

PLoS Genet 2020 10 14;16(10):e1009103. Epub 2020 Oct 14.

Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina, United States of America.

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1009103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588078PMC
October 2020

Defining the ATPome reveals cross-optimization of metabolic pathways.

Nat Commun 2020 08 28;11(1):4319. Epub 2020 Aug 28.

Gladstone Institute of Neurological Disease, San Francisco, CA, 94158, USA.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-18084-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455733PMC
August 2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

ACS Chem Biol 2020 08 12;15(8):2137-2153. Epub 2020 Aug 12.

Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, United States.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.0c00339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442712PMC
August 2020

CRISPR-based functional genomics for neurological disease.

Authors:
Martin Kampmann

Nat Rev Neurol 2020 Sep 8;16(9):465-480. Epub 2020 Jul 8.

Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, USA.

Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, as many lack disease-modifying treatments. A major reason for the absence of effective therapies is our limited understanding of the causative molecular and cellular mechanisms. Genome-wide association studies are providing a growing catalogue of disease-associated genetic variants, and the next challenge is to elucidate how these variants cause disease and to translate this understanding into therapies. This Review describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases. The bacterial CRISPR system can be used in experimental disease models to edit genomes and to control gene expression levels through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These genetic perturbations can be implemented in massively parallel genetic screens to evaluate the functional consequences for human cells. CRISPR screens are particularly powerful in combination with induced pluripotent stem cell technology, which enables the derivation of differentiated cell types, such as neurons and glia, and brain organoids from cells obtained from patients. Modelling of disease-associated changes in gene expression via CRISPRi and CRISPRa can pinpoint causal changes. In addition, genetic modifier screens can be used to elucidate disease mechanisms and causal determinants of cell type-selective vulnerability and to identify therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41582-020-0373-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484261PMC
September 2020

Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent.

Mol Cell 2020 07;79(1):191-198.e3

Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332992PMC
July 2020

CRISPR-based screens uncover determinants of immunotherapy response in multiple myeloma.

Blood Adv 2020 07;4(13):2899-2911

Institute for Neurodegenerative Diseases and.

Cancer cells commonly develop resistance to immunotherapy by loss of antigen expression. Combinatorial treatments that increase levels of the target antigen on the surface of cancer cells have the potential to restore efficacy to immunotherapy. Here, we use our CRISPR interference- and CRISPR activation-based functional genomics platform to systematically identify pathways controlling cell surface expression of the multiple myeloma immunotherapy antigen B-cell maturation antigen (BCMA). We discovered that pharmacologic inhibition of HDAC7 and the Sec61 complex increased cell surface BCMA, including in primary patient cells. Pharmacologic Sec61 inhibition enhanced the antimyeloma efficacy of a BCMA-targeted antibody-drug conjugate. A CRISPR interference chimeric antigen receptor T cells (CAR-T cells) coculture screen enabled us to identify both antigen-dependent and antigen-independent mechanisms controlling response of myeloma cells to BCMA-targeted CAR-T cells. Thus, our study shows the potential of CRISPR screens to uncover mechanisms controlling response of cancer cells to immunotherapy and to suggest potential combination therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2019001346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362346PMC
July 2020

LRP1 is a master regulator of tau uptake and spread.

Nature 2020 04 1;580(7803):381-385. Epub 2020 Apr 1.

Neuroscience Research Institute, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA.

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity. This observation and complementary experimental studies have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2156-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687380PMC
April 2020

Deep mutational scanning reveals the structural basis for α-synuclein activity.

Nat Chem Biol 2020 06 9;16(6):653-659. Epub 2020 Mar 9.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-0480-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339969PMC
June 2020

Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.

Nature 2020 03 4;579(7799):427-432. Epub 2020 Mar 4.

Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USA.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF4. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2078-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147832PMC
March 2020

Gene expression and cell identity controlled by anaphase-promoting complex.

Nature 2020 03 19;579(7797):136-140. Epub 2020 Feb 19.

Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA.

Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2034-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402266PMC
March 2020

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.

J Biol Chem 2019 12 2;294(50):18952-18966. Epub 2019 Oct 2.

Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94158

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.009432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916486PMC
December 2019

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses.

Mol Cell Proteomics 2019 12 30;18(12):2401-2417. Epub 2019 Sep 30.

Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany

Novel tick-borne phleboviruses in the family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also -, -, -, and In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.RA119.001631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885706PMC
December 2019

CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.

Neuron 2019 10 15;104(2):239-255.e12. Epub 2019 Aug 15.

Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Chan-Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2019.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813890PMC
October 2019

Ceapins block the unfolded protein response sensor ATF6α by inducing a neomorphic inter-organelle tether.

Elife 2019 05 31;8. Epub 2019 May 31.

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States.

The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.46595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588346PMC
May 2019

Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids.

Mol Cell 2019 04 4;74(1):32-44.e8. Epub 2019 Mar 4.

Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.01.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696670PMC
April 2019

Heterochromatin anomalies and double-stranded RNA accumulation underlie poly(PR) toxicity.

Science 2019 02;363(6428)

Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.

How hexanucleotide GGGGCC (GC) repeat expansions in cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded GC repeats. The expression of green fluorescent protein-conjugated (PR) (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of -associated FTD and ALS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aav2606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524780PMC
February 2019

Relapse-associated AURKB blunts the glucocorticoid sensitivity of B cell acute lymphoblastic leukemia.

Proc Natl Acad Sci U S A 2019 02 7;116(8):3052-3061. Epub 2019 Feb 7.

Department of Biochemistry, Holden Comprehensive Cancer Center, Carver College of Medicine, University of Iowa, Iowa City, IA 52242;

Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death. This complex selectively supports GC-induced expression of genes contributing to cell death. A metaanalysis of gene expression data from B-ALL patient specimens revealed that Aurora kinase B (AURKB), which restrains GC signaling by phosphorylating EHMT1-2, is overexpressed in relapsed B-ALL, suggesting it as a potential contributor to relapse. Inhibition of AURKB enhanced GC-induced expression of cell death genes, resulting in potentiation of GC cytotoxicity in cell lines and relapsed B-ALL patient samples. This function for AURKB is distinct from its canonical role in the cell cycle. These results show the utility of functional genomics in understanding mechanisms of resistance and rapidly identifying combination chemotherapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1816254116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386662PMC
February 2019

A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure.

PLoS Biol 2018 08 27;16(8):e2004624. Epub 2018 Aug 27.

Gladstone Institute of Neurological Disease, San Francisco, California, United States of America.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2004624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110572PMC
August 2018

CRISPR-based genetic interaction maps inform therapeutic strategies in cancer.

Transl Cancer Res 2018 Feb;7(Suppl 1):S61-S67

Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases and California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/tcr.2018.01.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107073PMC
February 2018

The Psychiatric Cell Map Initiative: A Convergent Systems Biological Approach to Illuminating Key Molecular Pathways in Neuropsychiatric Disorders.

Cell 2018 07;174(3):505-520

Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA; Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward-an initiative aimed at mapping the physical and genetic interaction networks of these conditions and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247911PMC
July 2018

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Biol Open 2018 Jul 23;7(7). Epub 2018 Jul 23.

Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biology (QBI), San Francisco 94158, USA

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/bio.036103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078352PMC
July 2018

Mapping the Genetic Landscape of Human Cells.

Cell 2018 08 19;174(4):953-967.e22. Epub 2018 Jul 19.

Department of Urology, University of California, San Francisco, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426455PMC
August 2018

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs).

Sci Rep 2018 04 23;8(1):6382. Epub 2018 Apr 23.

Neuroscience Research Institute, Department of Molecular Cellular Developmental Biology, University of California, Santa Barbara, CA, 93106, USA.

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-24904-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913225PMC
April 2018

Dual gene activation and knockout screen reveals directional dependencies in genetic networks.

Nat Biotechnol 2018 02 15;36(2):170-178. Epub 2018 Jan 15.

Department of Microbiology and Immunology, University of California San Francisco Diabetes Center, WM Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, California, USA.

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.4062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072461PMC
February 2018

CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine.

Authors:
Martin Kampmann

ACS Chem Biol 2018 02 24;13(2):406-416. Epub 2017 Oct 24.

Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases and California Institute for Quantitative Biomedical Research, University of California , San Francisco, California 94158, United States.

Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidating gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases. The focus of this review is the use of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for genetic screens in mammalian cells. We introduce the underlying technology and present different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes. Combinatorial screens, in which large numbers of gene pairs are targeted to construct genetic interaction maps, reveal pathway relationships and protein complexes. We compare and contrast CRISPRi and CRISPRa with alternative technologies, including RNA interference (RNAi) and CRISPR nuclease-based screens. Finally, we highlight challenges and opportunities ahead.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.7b00657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886776PMC
February 2018

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.

Mol Cell 2017 Oct;68(1):210-223.e6

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640507PMC
October 2017