A CRISPR screen in intestinal epithelial cells identifies novel factors for polarity and apical transport.

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.

The SKP2-p27 axis defines susceptibility to cell death upon CHK1 inhibition.

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under-replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B-Cell CLL/Lymphoma 2 (BCL2)-regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53-deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome-wide CRISPR/Cas9-based loss-of-function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F-box protein S-phase Kinase-Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin-dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1-S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1-inhibitor treatment and reduced cell death in S-phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1-inhibitor treatment.

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Checkpoint kinase 1 is essential for normal B cell development and lymphomagenesis.

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells and studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor. Here, we report on the key role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1 inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA. Our findings define CHK1 as essential in B-cell development and potent target to treat blood cancer.

The miR-15 family reinforces the transition from proliferation to differentiation in pre-B cells.

Precursor B lymphocytes expand upon expression of a pre-B cell receptor (pre-BCR), but then transit into a resting state in which immunoglobulin light chain gene recombination is initiated. This bi-phasic sequence is orchestrated by the IL-7 receptor (IL-7R) and pre-BCR signaling, respectively, but little is known about microRNAs fine-tuning these events. Here, we show that pre-B cells lacking miR-15 family functions exhibit prolonged proliferation due to aberrant expression of the target genes cyclin E1 and D3. As a consequence, they fail to trigger the transcriptional reprogramming normally accompanying their differentiation, resulting in a developmental block at the pre-B cell stage. Intriguingly, our data indicate that the miR-15 family is suppressed by both IL-7R and pre-BCR signaling, suggesting it is actively integrated into the regulatory circuits of developing B cells. These findings identify the miR-15 family as a novel element required to promote the switch from pre-B cell proliferation to differentiation.