Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most ...knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.
In the 12 years since the process of RNA interference (RNAi) was first discovered, great progress has been made in understanding its mechanism and exploiting its ability to silence gene expression to ...study gene function at a genome-wide level. Its extensive use as a screening method has yielded many published lists of genes that play novel roles in higher eukaryotes. However, the usefulness of this information is potentially limited by the occurrence of unintended off-target effects. Here we review the potential causes of off-target effects and the impact of this phenomenon in interpreting the results of high-throughput RNAi screens. In addition to targeting the intended gene product, artificial short interfering RNAs (siRNAs) can produce off-target effects by down-regulating the expression of multiple mRNAs through microRNA-like targeting of the 3' untranslated region. We examine why this phenomenon can produce high hit rates in siRNA screens and why independent validation of screening results is critical for the approach to yield new biological insights.
Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of ...cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.
Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase ...that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome. Because blocking mitotic exit is an effective approach for inducing tumour cell death, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box). Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C-Cdc20 interaction. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine.
Because off-target effects hamper interpretation and validation of RNAi screen data, we developed a bioinformatics method, genome-wide enrichment of seed sequence matches (GESS), to identify ...candidate off-targeted transcripts in primary screening data. GESS analysis revealed a prominent off-targeted transcript in several screens, including MAD2 (MAD2L1) in a screen for genes required for the spindle assembly checkpoint. GESS analysis results can enhance the validation rate in RNAi screens.
The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we ...identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.
Abstract
Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many ...transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.
Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the ...anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.
► TAME is a small molecule that inhibits APC activation by preventing Cdc20 binding ► A cell-permeable prodrug (proTAME) induces mitotic arrest and cell death ► APC-dependent proteolysis is required for spindle-assembly checkpoint inactivation ► ProTAME exploits mutual antagonism between the SAC and APC to block mitotic exit
A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg ...loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Display omitted
•Genome-wide CRISPR screen identifies host factors regulating HBsAg expression•ZCCHC14 and TENT4A/B stabilize HBsAg expression through RNA tailing•ZCCHC14 forms a complex with TENT4B and the HBV PRE•RG7834 inhibits the enzymatic activity of TENT4A/B within the ZCCHC14-HBV PRE complex
Hyrina et al. employ a non-biased functional CRISPR screening approach to identify host factors regulating HBsAg expression as well as those targeted by RG7834, a HBsAg inhibitor. The screen highlighted over 60 genes and identified a mechanism by which ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing.
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we ...combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
PDF
