Biology subjects

Virshup, D. M.

Publications and source records attributed to Virshup, D. M..

14 recordsLinked to original sources

bioRxiv2025

Identification of Wnt regulated genes that are repressed by, or independent of, β-catenin

Wnt signaling regulates metazoan development and homeostasis, in part by {beta}-catenin dependent activation and repression of a large number of genes. However, Wnt signaling also regulates genes independent of {beta}-catenin, genes that are less well characterized. In this study, using a pan-Wnt inhibitor we performed a comprehensive transcriptome analysis in a Wnt-addicted orthotopic cancer model to delineate the {beta}-catenin-dependent and independent arms of Wnt signaling. We find that while a large percentage of Wnt-regulated genes are regulated by {beta}-catenin, ten percent of these genes are regulated independent of {beta}-catenin. Interestingly, a large proportion of these {beta}-catenin independent genes are Wnt-repressed. Among the {beta}-catenin dependent genes, more than half are repressed by {beta}-catenin. We used this dataset to investigate the mechanisms by which Wnt/{beta}-catenin signaling represses gene expression, revealing the role of a cis-regulatory motif, the negative regulatory element (NRE). The NRE motif is enriched in the promoters of {beta}-catenin repressed genes and is required for their repression. This provides a comprehensive analysis of the {beta}-catenin independent arm of the Wnt signaling pathway in a cancer model and illustrates how a cis-regulatory grammar can determine Wnt-dependent gene activation versus repression.

cell biology↗

bioRxiv2025

Markovian State Models uncover Casein Kinase 1 dynamics that govern circadian period

Circadian rhythms in mammals are tightly regulated through phosphorylation of Period (PER) proteins by Casein Kinase 1 (CK1, subtypes {delta} and {varepsilon}). CK1 acts on at least two different regions of PER with opposing effects: phosphorylation of phosphodegron (pD) regions leads to PER degradation, while phosphorylation of the Familial Advanced Sleep Phase (FASP) region leads to PER stabilization. To investigate how substrate selectivity is encoded by the conformational dynamics of CK1, we performed a large set of independent molecular dynamics (MD) simulations of wildtype CK1 and the tau mutant (R178C) that biases kinase activity toward a pD. We used Markovian State Models (MSMs) to integrate the simulations into a single model of the conformational landscape of CK1 and used Gaussian accelerated molecular dynamics (GaMD) to build the first molecular model of CK1 and the unphosphorylated FASP motif. Together, these findings provide a mechanistic view of CK1, establishing how the activation loop acts as a key molecular switch to control substrate selectivity. We show that the tau mutant favors an alternative conformation of the activation loop and significantly accelerates the dynamics of CK1. This reshapes the binding cleft in a way that impairs FASP binding and would ultimately lead to PER destabilization and shorter circadian periods. Finally, we identified an allosteric pocket that could be targeted to bias this molecular switch. Our integrated approach offers a detailed model of CK1s conformational landscape and its relevance to normal, mutant, and druggable circadian timekeeping. Statement of SignificanceDisruption of circadian rhythms alters the temporal orchestration of vital cellular processes and increases the propensity for sleep disorders, metabolic disease, and cancer. Circadian rhythms are generated by a vast gene expression program controlled at the cellular level by a molecular clock comprised of dedicated clock proteins. Amongst the essential protein characters is Casein kinase 1 (CK1), which acts on multiple clock protein substrates. A delicate balance of CK1 activity on these substrates is crucial for proper circadian timekeeping, highlighting CK1 as a promising drug target to tune clock timing. This work aims to identify the conformational landscape of CK1 that underlies its substrate specificity and provide molecular insight for pharmacologic development that could modulate CK1 function for those suffering from clock-related syndromes.

biophysics↗

bioRxiv2023

The cholesterol biosynthesis enzyme FAXDC2 couples Wnt/beta-catenin to RTK/MAPK signaling

Wnts, cholesterol, and MAPK signaling are essential for development and adult homeostasis. Here we report for the first time that fatty acid hydroxylase domain containing 2 (FAXDC2), a previously uncharacterized enzyme, functions as a methyl sterol oxidase catalyzing C4 demethylation in the Kandutsch-Russell branch of the cholesterol biosynthesis pathway. FAXDC2, a paralog of MSMO1, regulates the abundance of specific C4-methyl sterols lophenol and dihydro-TMAS. Highlighting its clinical relevance, FAXDC2 is repressed in Wnt/{beta}-catenin high cancer xenografts, in a mouse genetic model of Wnt activation, and in human colorectal cancers. Moreover, in primary human colorectal cancers, the sterol lophenol, regulated by FAXDC2, accumulates in the cancerous tissues and not in adjacent normal tissues. FAXDC2 links Wnts to RTK/MAPK signaling. Wnt inhibition drives increased recycling of RTKs and activation of the MAPK pathway, and this requires FAXDC2. Blocking Wnt signaling in Wnt-high cancers causes both differentiation and senescence; and this is prevented by knockout of FAXDC2. Our data shows the integration of three ancient pathways, Wnts, cholesterol synthesis, and RTK/MAPK signaling, in cellular proliferation and differentiation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/571793v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@6608f2org.highwire.dtl.DTLVardef@d5566eorg.highwire.dtl.DTLVardef@1d889aorg.highwire.dtl.DTLVardef@f37411_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability

The duration of the transcription-repression cycles that give rise to mammalian circadian rhythms is largely determined by the stability of the PERIOD protein, the rate-limiting components of the molecular clock. The degradation of PERs is tightly regulated by multisite phosphorylation by Casein Kinase 1 (CK1{delta}/{varepsilon}). In this phosphoswitch, phosphorylation of a PER2 degron (Degron 2, D2) causes degradation, while phosphorylation of the PER2 Familial Advanced Sleep Phase (FASP) domain blocks CK1 activity on the degron, stabilizing PER2. However, this model and many other studies of PER2 degradation do not include the second degron of PER2 that is conserved in PER1, termed Degron 1, D1. We examined how these two degrons contribute to PER2 stability, affect the balance of the phosphoswitch, and how they are differentiated by CK1. Using PER2-luciferase fusions and real-time luminometry, we investigated the contribution of both D2 and of CK1-PER2 binding. We find that D1, like D2, is a substrate of CK1 but that D1 plays only a backup role in PER2 degradation. Notably, CK1 bound to a PER1:PER2 dimer protein can phosphorylate PER1 D1 in trans. This scaffolded phosphorylation provides additional levels of control to PER stability and circadian rhythms.

cell biology↗

bioRxiv2023

Recurrent FBXW7 mutations bypass Wnt/β-catenin addiction in cancer

Pathologic Wnt/{beta}-catenin signaling drives various cancers, leading to multiple approaches to drug this pathway. Appropriate patient selection can maximize success of these interventions. Wnt ligand addiction is a druggable vulnerability in RNF43-mutant/RSPO-fusion cancers. However, pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, has shown mixed therapeutic responses, possibly due to tumor heterogeneity. Here we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors, and FBXW7 mutations cause intrinsic resistance to anti-Wnt therapies. Mechanistically, inactivation of FBXW7 stabilizes multiple oncoproteins including Cyclin E and MYC, and antagonizes the cytostatic effect of Wnt inhibitors. Moreover, although FBXW7 mutations do not mitigate {beta}-catenin degradation upon Wnt inhibition, FBXW7-mutant RNF43-mutant/RSPO-fusion cancers instead lose dependence on {beta}-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/{beta}-catenin-independent tumors are susceptible to multi-CDK inhibition by dinaciclib. An in depth understanding of primary resistance to anti-Wnt/{beta}-catenin therapies allows for more appropriate patient selection and use of alternative mechanism-based therapies.

cancer biology↗

bioRxiv2023

Isoform-specific C-terminal phosphorylation drives autoinhibition of Casein Kinase 1

Casein kinase 1 {delta} (CK1{delta}) controls essential biological processes including circadian rhythms and Wnt signaling, but how its activity is regulated is not well understood. CK1{delta} is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, {delta}1 and {delta}2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C-termini (XCT), but with marked changes in potential phosphorylation sites. Here we test if the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and HDX-MS, we show that the {delta}1 XCT is preferentially phosphorylated by the kinase and the {delta}1 tail makes more extensive interactions across the kinase domain. Mutation of {delta}1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. {delta}1 autoinhibition relies on conserved anion binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1{delta}. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase. SignificanceSubtle control of kinase activity is critical to physiologic modulation of multiple physiological processes including circadian rhythms. CK1{delta} and the closely related CK1{varepsilon} regulate circadian rhythms by phosphorylation of PER2, but how kinase activity itself is controlled is not clear. Building on the prior observation that two splice isoforms of CK1{delta} regulate the clock differently, we show that the difference maps to three phosphorylation sites in the variably spliced region (XCT) that cause feedback inhibition of the kinase domain. More broadly, the data suggest a general model where CK1 activity on diverse substrates can be controlled by signaling pathways that alter tail phosphorylation. These inhibitory phosphorylation sites could also be targets for new therapeutic interventions.

biochemistry↗

bioRxiv2022

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period

PERIOD (PER) and Casein Kinase 1{delta} regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1{delta} phosphorylation of the Familial Advanced Sleep Phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1{delta}. Co-crystal structures in conjunction with accelerated molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1{delta}. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortens circadian period in human cells. We found that Drosophila PER also regulates CK1{delta} via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

biochemistry↗

bioRxiv2021

Structural model of PORCN illuminates disease-associated variants and drug binding sites

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Here, based on homology to mammalian MBOAT proteins we develop and validate a molecular structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model provides mechanistic insights into PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

molecular biology↗

bioRxiv2020

Vangl2 regulates the dynamics of Wnt cytonemes in vertebrates

The Wnt signalling network regulates cell proliferation and cell differentiation as well as migration and polarity in development of multicellular organisms. However, it is still unclear how distribution of Wnt ligands is precisely controlled to fulfil all of these different functions. Here, we show that the four-pass transmembrane protein Vangl2 occupies a central role in determining the distribution of Wnt by cytonemes in vertebrate tissue. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, activation of Vangl2 leads to the generation of fewer but extremely long cytonemes, which start to branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/{beta}-catenin signalling in the surrounding cells. Concordantly, inhibition of Vangl2 function leads to the formation of shorter cytonemes and reduced paracrine Wnt/{beta}-catenin signal activation. A mathematical model simulating the observed Vangl2 functions on cytonemes in zebrafish gastrulation predicts an anterior shift of the morphogenetic signalling gradient, altered tissue patterning, and a loss of the sharpness of tissue domains. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we show that Vangl2 - a core member of the PCP signalling component - is fundamental to paracrine Wnt/{beta}-catenin signalling by controlling cytoneme behaviour in vertebrate development and tissue homeostasis.

developmental biology↗

bioRxiv2020

The functional landscape of patient derived RNF43 mutations predicts Wnt inhibitor sensitivity

A subset of Wnt-addicted cancers are sensitive to targeted therapies that block Wnt secretion or receptor engagement. RNF43 loss-of-function mutations that increase cell surface Wnt receptor abundance cause sensitivity to Wnt inhibitors. However, it is not clear which of the clinically identified RNF43 mutations affect its function in vivo. We assayed 90 missense and 45 truncating RNF43 mutations found in human cancers, using a combination of cell-based reporter assays, genome editing, flow cytometry and immunofluorescence microscopy. Patent-derived xenograft (PDX) models with C-terminal truncating RNF43 mutations were tested for Wnt inhibitor sensitivity. We find that five common germline variants of RNF43 have wild-type activity. The majority of cancer-associated missense mutations in the RING and PA domains are either loss of function or hyperactivating. Hyperactivating mutants appear to function through formation of inactive dimers with endogenous RNF43 and/or ZNRF3. C-terminal truncation mutants including the common G659fs mutant, have discordant behavior in in vitro versus in vivo assays. PDXs and cell lines with C-terminal truncations show increased cell surface FZD, Wnt/{beta}-catenin signaling and are responsive to PORCN inhibition in vivo, providing clear evidence of RNF43 loss of function. In conclusion, RNF43 nonsense and frameshift mutations, including those in the C-terminal domain, and specific missense mutations in RING and PA are loss of function and predict response to upstream Wnt inhibitors in microsatellite stable cancers. This study expands the landscape of actionable RNF43 mutations, potentially extending the benefit of these therapies to additional patients. Statement of SignificanceLoss of function RNF43 mutations, first described in pancreatic cancers, drive progression of multiple cancers by increasing cellular sensitivity to Wnt ligands. These cancers are therefore uniquely sensitive to agents such as PORCN inhibitors that block Wnt production. As the PORCN inhibitors and other upstream inhibitors advance into clinical trials it is important to identify the right patients to treat with these upstream Wnt inhibitors. Hence a detailed map of mutations that are actionable is required. Here we systematically examined a spectrum of 135 patient-derived RNF43 mutations from multiple cancers. Using cell-based reporter assays, genome editing and patient-derived xenografts, we identify rules to guide patient selection. MSS cancers with either truncating mutations anywhere in the gene, including C-terminal truncations around the G659 position, or point mutations in well-defined functional domains, are likely to have RNF43 loss of function and hence a response to therapy.

cancer biology↗

bioRxiv2020

Wnt-regulated lncRNA discovery enhanced by in vivo identification and CRISPRi functional validation

BackgroundWnt signaling is an evolutionarily conserved developmental pathway that is frequently hyperactivated in cancer. While multiple protein-coding genes regulated by Wnt signaling are known, the functional lncRNAs regulated by Wnt signaling have not been systematically characterized. ResultsWe comprehensively mapped lncRNAs from an orthotopic Wnt-addicted pancreatic cancer model, identifying 3,633 lncRNAs, of which 1,503 were regulated by Wnt signaling. We found lncRNAs were much more sensitive to changes in Wnt signaling in xenografts than in cultured cells. To functionally validate Wnt-regulated lncRNAs, we performed CRISPRi screens to assess their role in cancer cell proliferation. Consistent with previous genome-wide lncRNA CRISPRi screens, around 1% (13/1,503) of the Wnt-regulated lncRNAs could modify cancer cell growth in vitro. This included CCAT1 and LINC00263, previously reported to regulate cancer growth. Using an in vivo CRISPRi screen, we doubled the discovery rate, identifying twice as many Wnt-regulated lncRNAs (25/1,503) that had a functional effect on cancer cell growth. ConclusionsOur study demonstrates the value of studying lncRNA functions in vivo, provides a valuable resource of lncRNAs regulated by Wnt signaling and establishes a framework for systematic discovery of functional lncRNAs.

genomics↗

bioRxiv2019

Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase {beta}-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 mRNA levels were minimally affected. Nuclear PER1, CRY1 and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.

molecular biology↗

bioRxiv2019

Casein kinase 1 dynamics underlie the PER2 circadian phosphoswitch

Post-translational control of PERIOD stability by Casein Kinase 1{delta} and {varepsilon} (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the activation loop to define substrate selectivity on mammalian PER2, thereby directly regulating its stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.

biochemistry↗

bioRxiv2018

Visualizing Wnt secretion from endoplasmic reticulum to filopodia

Wnts are a family of secreted palmitoleated glycoproteins that play a key role in cell to cell communications during development and regulate stem cell compartments in adults. Wnt receptors, downstream signaling cascades and target pathways have been extensively studied while less is known about how Wnts are secreted and move from producing cells to receiving cells. We used the synchronization system called Retention Using Selective Hook (RUSH) to study Wnt trafficking from endoplasmic reticulum to Golgi and then to plasma membrane and filopodia in real time. Consistent with prior studies, inhibition of porcupine (PORCN) or knockout of Wntless (WLS) blocked Wnt exit from the ER. Indeed, WLS was rate-limiting for Wnt ER exit. Wnt-containing vesicles paused at sub-cortical regions of the plasma membrane before exiting the cell. Wnt-containing vesicles were transported to adjacent cells associated with filopodia. Increasing the number of filopodia by expression of LGR5 in the producing cell increased the ability of a cell to send a Wnt signal. The RUSH system is a powerful tool to provide new insights into the Wnt secretory pathway.

cell biology↗