CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation.

The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.

Stereoselective fatty acylation is essential for the release of lipidated WNT proteins from the acyltransferase Porcupine (PORCN).

The maintenance of adult animal tissues depends upon highly conserved intercellular signaling molecules that include the secreted WNT proteins. Although it is generally accepted that lipidation of WNTs by the acyltransferase Porcupine (PORCN) and their subsequent recognition by the Wntless (WLS) protein is essential for their cellular secretion, the molecular understanding of this process remains limited. Using structurally diverse fatty acyl donor analogs and mouse embryonic fibroblasts expressing PORCN protein from different metazoan phyla, we demonstrate here that PORCN active-site features, which are conserved across the animal kingdom, enforce cis-Δ9 fatty acylation of WNTs. Aberrant acylation of a WNT with an exogenously supplied trans-Δ9 fatty acid induced the accumulation of WNT-PORCN complexes, suggesting that the fatty acyl species is critical for the extrication of lipidated WNTs from PORCN. Our findings reveal a previously unrecognized fatty acyl-selective checkpoint in the manufacturing of a lipoprotein that forms a basis for WNT signaling sensitivity to trans fats and to PORCN inhibitors in clinical development.

Installation of a cancer promoting WNT/SIX1 signaling axis by the oncofusion protein MLL-AF9.

BACKGROUND: Chromosomal translocation-induced expression of the chromatin modifying oncofusion protein MLL-AF9 promotes acute myelocytic leukemia (AML). Whereas WNT/ß-catenin signaling has previously been shown to support MLL-AF9-driven leukemogenesis, the mechanism underlying this relationship remains unclear. METHODS: We used two novel small molecules targeting WNT signaling as well as a genetically modified mouse model that allow targeted deletion of the WNT protein chaperone Wntless (WLS) to evaluate the role of WNT signaling in AML progression. ATAC-seq and transcriptome profiling were deployed to understand the cellular consequences of disrupting a WNT signaling in leukemic initiating cells (LICs). FINDINGS: We identified Six1 to be a WNT-controlled target gene in MLL-AF9-transformed leukemic initiating cells (LICs). MLL-AF9 alters the accessibility of Six1 DNA to the transcriptional effector TCF7L2, a transducer of WNT/ß-catenin gene expression changes. Disruption of WNT/SIX1 signaling using inhibitors of the Wnt signaling delays the development of AML. INTERPRETATION: By rendering TCF/LEF-binding elements controlling Six1 accessible to TCF7L2, MLL-AF9 promotes WNT/ß-catenin-dependent growth of LICs. Small molecules disrupting WNT/ß-catenin signaling block Six1 expression thereby disrupting leukemia driven by MLL fusion proteins.

Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor.

Direct interactions between pro- and anti-apoptotic BCL-2 family members form the basis of cell death decision-making at the outer mitochondrial membrane (OMM). Here we report that three anti-apoptotic BCL-2 proteins (MCL-1, BCL-2 and BCL-XL) found untethered from the OMM function as transcriptional regulators of a prosurvival and growth program. Anti-apoptotic BCL-2 proteins engage a BCL-2 homology (BH) domain sequence found in SUFU (suppressor of fused), a tumour suppressor and antagonist of the GLI DNA-binding proteins. BCL-2 proteins directly promote SUFU turnover, inhibit SUFU-GLI interaction, and induce the expression of the GLI target genes BCL-2, MCL-1 and BCL-XL. Anti-apoptotic BCL-2 protein/SUFU feedforward signalling promotes cancer cell survival and growth, and can be disabled with BH3 mimetics-small molecules that target anti-apoptotic BCL-2 proteins. Our findings delineate a chemical strategy for countering drug resistance in GLI-associated tumours and reveal unanticipated functions for BCL-2 proteins as transcriptional regulators.

Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.

We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.

Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making.

The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms.

The development of highly potent inhibitors for porcupine.

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Diverse chemical scaffolds support direct inhibition of the membrane-bound O-acyltransferase porcupine.

Secreted Wnt proteins constitute one of the largest families of intercellular signaling molecules in vertebrates with essential roles in embryonic development and adult tissue homeostasis. The functional redundancy of Wnt genes and the many forms of cellular responses they elicit, including some utilizing the transcriptional co-activator ß-catenin, has limited the ability of classical genetic strategies to uncover their roles in vivo. We had previously identified a chemical compound class termed Inhibitor of Wnt Production (or IWP) that targets Porcupine (Porcn), an acyltransferase catalyzing the addition of fatty acid adducts onto Wnt proteins. Here we demonstrate that diverse chemical structures are able to inhibit Porcn by targeting its putative active site. When deployed in concert with small molecules that modulate the activity of Tankyrase enzymes and glycogen synthase kinase 3 ß (GSK3ß), additional transducers of Wnt/ß-catenin signaling, the IWP compounds reveal an essential role for Wnt protein fatty acylation in eliciting ß-catenin-dependent and -independent forms of Wnt signaling during zebrafish development. This collection of small molecules facilitates rapid dissection of Wnt gene function in vivo by limiting the influence of redundant Wnt gene functions on phenotypic outcomes and enables temporal manipulation of Wnt-mediated signaling in vertebrates.

Conformational adaptation of apolipoprotein A-I to discretely sized phospholipid complexes.

The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96 A diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 A) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS-PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of two apoA-I molecules bound to approximately 150 molecules of POPC and suggest that the protein hydrophobic interactions at the N- and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N- and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.