ABSTRACT
N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.
Subject(s)
Congenital Disorders of Glycosylation , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Gene Expression Regulation , Humans , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/deficiency , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer-target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genome, Human , RNA-Seq/methods , Single-Cell Analysis/methods , Transcriptome/genetics , HumansABSTRACT
N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.
Subject(s)
Endoplasmic Reticulum , Gene Expression Regulation , Endoplasmic Reticulum/metabolism , Humans , K562 Cells , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Proteasome Endopeptidase Complex/metabolismABSTRACT
The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-ß-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency.
Subject(s)
Congenital Disorders of Glycosylation/metabolism , Lipid Metabolism/physiology , Liver/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/deficiency , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Stress, Physiological/physiology , Animals , Cell Line , Cytoplasm/metabolism , Diet , Disease Models, Animal , Endoplasmic Reticulum-Associated Degradation/physiology , Female , Fructose/metabolism , Glycosylation , Hepatocytes/metabolism , Male , Mice , PhenotypeABSTRACT
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Knockout Techniques , Cell Cycle Proteins/genetics , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Exons , Humans , Mutation , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Transcription Factors/geneticsABSTRACT
Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.
ABSTRACT
In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.
ABSTRACT
Hundreds of transcript isoforms with varying boundaries and alternative regulatory signals are transcribed from the genome, even in a genetically homogeneous population of cells. To study this transcriptional heterogeneity, we developed transcript isoform sequencing (TIF-seq), a method that allows the genome-wide profiling of full-length transcript isoforms defined by their exact 5' and 3' boundaries. TIF-seq entails the generation of full-length cDNA libraries, followed by their circularization and the sequencing of the junction fragments spanning the 5' and 3' transcript ends. By determining the respective co-occurrence of start and end sites of individual transcript molecules, TIF-seq can distinguish variations that conventional approaches for mapping single ends cannot, such as short abortive transcripts, bicistronic messages and overlapping transcripts that differ in lengths. The TIF-seq protocol we describe here can be applied to any eukaryotic organism (e.g., yeast, human), and it requires 6-10 d for generating TIF-seq libraries, 10 d for sequencing and 2-3 d for analysis.
Subject(s)
Protein Isoforms/chemistry , RNA, Messenger/chemistry , Saccharomyces cerevisiae/genetics , Sequence Analysis, RNA/methods , Genetic Variation , Genome , Genomics , Protein Isoforms/genetics , RNA, Messenger/geneticsABSTRACT
Unraveling the molecular processes that lead from genotype to phenotype is crucial for the understanding and effective treatment of genetic diseases. Knowledge of the causative genetic defect most often does not enable treatment; therefore, causal intermediates between genotype and phenotype constitute valuable candidates for molecular intervention points that can be therapeutically targeted. Mapping genetic determinants of gene expression levels (also known as expression quantitative trait loci or eQTL studies) is frequently used for this purpose, yet distinguishing causation from correlation remains a significant challenge. Here, we address this challenge using extensive, multi-environment gene expression and fitness profiling of hundreds of genetically diverse yeast strains, in order to identify truly causal intermediate genes that condition fitness in a given environment. Using functional genomics assays, we show that the predictive power of eQTL studies for inferring causal intermediate genes is poor unless performed across multiple environments. Surprisingly, although the effects of genotype on fitness depended strongly on environment, causal intermediates could be most reliably predicted from genetic effects on expression present in all environments. Our results indicate a mechanism explaining this apparent paradox, whereby immediate molecular consequences of genetic variation are shared across environments, and environment-dependent phenotypic effects result from downstream integration of environmental signals. We developed a statistical model to predict causal intermediates that leverages this insight, yielding over 400 transcripts, for the majority of which we experimentally validated their role in conditioning fitness. Our findings have implications for the design and analysis of clinical omics studies aimed at discovering personalized targets for molecular intervention, suggesting that inferring causation in a single cellular context can benefit from molecular profiling in multiple contexts.
Subject(s)
Gene Expression , Gene-Environment Interaction , Metabolic Networks and Pathways/genetics , Quantitative Trait Loci/genetics , Bayes Theorem , Environment , Genotype , Humans , Models, Statistical , Phenotype , Saccharomyces cerevisiae/geneticsABSTRACT
BACKGROUND: Post-puberty deterioration of kidneys is more rapid in males than in females. To reveal the underlying molecular mechanisms for this difference, we analyzed gender-dependent gene expression in kidneys of three groups of 36 day-old rats. RESULTS: The number of genes exhibiting gender-dependent expression was highly influenced by the genetic background of the rat group examined. 373, 288 and 79 genes showed differential gene expression between males and females (p = 0.001) in US, Mhm and Mhm*BN rats, respectively. Of all gender dependently expressed genes, only 39 genes were differentially expressed in all tested groups and the direction of expression change was the same for those genes for all groups. The gene expression profile suggests higher metabolic and transport activities, enhanced cell proliferation, elevated oxidative stress, and altered vascular biology in males. Furthermore, elevated levels of superoxide anion (two- to three-fold) in males compared to females were detected at early puberty, but neither at pre-puberty nor at late puberty/early adulthood. CONCLUSION: Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.