A Genetic Screen Identifies a Critical Role for the WDR81-WDR91 Complex in the Trafficking and Degradation of Tetherin.

Tetherin (BST2/CD317) is a viral restriction factor that anchors enveloped viruses to host cells and limits viral spread. The HIV-1 Vpu accessory protein counteracts tetherin by decreasing its cell surface expression and targeting it for ubiquitin-dependent endolysosomal degradation. Although the Vpu-mediated downregulation of tetherin has been extensively studied, the molecular details are not completely elucidated. We therefore used a forward genetic screen in human haploid KBM7 cells to identify novel genes required for tetherin trafficking. Our screen identified WDR81 as a novel gene required for tetherin trafficking and degradation in both the presence and absence of Vpu. WDR81 is a BEACH-domain containing protein that is also required for the degradation of EGF-stimulated epidermal growth factor receptor (EGFR) and functions in a complex with the WDR91 protein. In the absence of WDR81 the endolysosomal compartment appears swollen, with enlarged early and late endosomes and reduced delivery of endocytosed dextran to cathepsin-active lysosomes. Our data suggest a role for the WDR81-WDR91 complex in the fusion of endolysosomal compartments and the absence of WDR81 leads to impaired receptor trafficking and degradation.

A human haploid gene trap collection to study lncRNAs with unusual RNA biology.

Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized LOC100288798 lncRNA as a model to answer this question. Using public RNA-seq data we show that LOC100288798 is ubiquitously expressed, but inefficiently spliced. The minor spliced LOC100288798 isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that LOC100288798 RNA biology differs markedly from typical mRNAs. De novo assembly from RNA-seq data suggests that LOC100288798 extends 289kb beyond its annotated 3' end and overlaps the downstream SLC38A4 gene. Three cell lines with independent gene trap insertions in LOC100288798 were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon LOC100288798 truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized LOC100288798 as a potential gene regulator.

GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.

Distinct Mechanisms of Resistance to a CENP-E Inhibitor Emerge in Near-Haploid and Diploid Cancer Cells.

Aberrant chromosome numbers in cancer cells may impose distinct constraints on the emergence of drug resistance-a major factor limiting the long-term efficacy of molecularly targeted therapeutics. However, for most anticancer drugs we lack analyses of drug-resistance mechanisms in cells with different karyotypes. Here, we focus on GSK923295, a mitotic kinesin CENP-E inhibitor that was evaluated in clinical trials as a cancer therapeutic. We performed unbiased selections to isolate inhibitor-resistant clones in diploid and near-haploid cancer cell lines. In diploid cells we identified single-point mutations that can suppress inhibitor binding. In contrast,transcriptome analyses revealed that the C-terminus of CENP-E was disrupted in GSK923295-resistant near-haploid cells. While chemical inhibition of CENP-E is toxic to near-haploid cells, knockout of the CENPE gene does not suppress haploid cell proliferation, suggesting that deletion of the CENP-E C-terminus can confer resistance to GSK923295. Together, these findings indicate that different chromosome copy numbers in cells can alter epistatic dependencies and lead to distinct modes of chemotype-specific resistance.

Identification of Genes That Modulate Susceptibility to Formaldehyde and Imatinib by Functional Genomic Screening in Human Haploid KBM7 Cells.

Though current functional genomic screening systems are useful for investigating human susceptibility to chemical toxicity, they have limitations. Well-established, high-throughput yeast mutant screens identify only evolutionarily conserved processes. RNA interference can be applied in human cells but is limited by incomplete gene knockout and off-target effects. Human haploid cell screening is advantageous as it requires knockdown of only a single copy of each gene. A human haploid cell mutant library (KBM7-Mu), derived from a chronic myeloid leukemia (CML) patient, was recently developed and has been used to identify genes that modulate sensitivity to infectious agents and pharmaceutical drugs. Here, we sought to improve the KBM7-Mu screening process to enable efficient screening of environmental chemicals. We developed a semi-solid medium based screening approach that cultures individual mutant colonies from chemically resistant cells, faster (by 2-3 weeks) and with less labor than the original liquid medium-based approach. As proof of principle, we identified genetic mutants that confer resistance to the carcinogen formaldehyde (FA, 12 genes, 18 hits) and the CML chemotherapeutic agent imatinib (6 genes, 13 hits). Validation experiments conducted on KBM7 mutants lacking each of the 18 genes confirmed resistance of 6 FA mutants (CTC1, FCRLA, GOT1, LPR5, M1AP, and MAP2K5) and 1 imatinib-resistant mutant (LYRM9). Despite the improvements to the method, it remains technically challenging to limit false positive findings. Nonetheless, our findings demonstrate the broad applicability of this optimized haploid approach to screen toxic chemicals to identify novel susceptibility genes and gain insight into potential mechanisms of toxicity.

Functional genomic screening approaches in mechanistic toxicology and potential future applications of CRISPR-Cas9.

Characterizing variability in the extent and nature of responses to environmental exposures is a critical aspect of human health risk assessment. Chemical toxicants act by many different mechanisms, however, and the genes involved in adverse outcome pathways (AOPs) and AOP networks are not yet characterized. Functional genomic approaches can reveal both toxicity pathways and susceptibility genes, through knockdown or knockout of all non-essential genes in a cell of interest, and identification of genes associated with a toxicity phenotype following toxicant exposure. Screening approaches in yeast and human near-haploid leukemic KBM7 cells have identified roles for genes and pathways involved in response to many toxicants but are limited by partial homology among yeast and human genes and limited relevance to normal diploid cells. RNA interference (RNAi) suppresses mRNA expression level but is limited by off-target effects (OTEs) and incomplete knockdown. The recently developed gene editing approach called clustered regularly interspaced short palindrome repeats-associated nuclease (CRISPR)-Cas9, can precisely knock-out most regions of the genome at the DNA level with fewer OTEs than RNAi, in multiple human cell types, thus overcoming the limitations of the other approaches. It has been used to identify genes involved in the response to chemical and microbial toxicants in several human cell types and could readily be extended to the systematic screening of large numbers of environmental chemicals. CRISPR-Cas9 can also repress and activate gene expression, including that of non-coding RNA, with near-saturation, thus offering the potential to more fully characterize AOPs and AOP networks. Finally, CRISPR-Cas9 can generate complex animal models in which to conduct preclinical toxicity testing at the level of individual genotypes or haplotypes. Therefore, CRISPR-Cas9 is a powerful and flexible functional genomic screening approach that can be harnessed to provide unprecedented mechanistic insight in the field of modern toxicology.

Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study.

INTRODUCTION: High-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. METHODS: KBM7-mu cells were exposed to 200 µM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h of treatment. After a 2-3 week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. RESULTS: We identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. DISCUSSION: The KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity.