FOXO3a and beta-catenin transcriptional profile in human colon cancer cell line DLD1
Ontology highlight
ABSTRACT: We established a model system in human DLD1 colon cancer cells to study the transcriptional crosstalk between FOXO3A and beta-Catenin. Thereby, translocation to the nucleus of a AKT-insensitive mutant (T32A, S253A, S315A) of human FOXO3A fused to the ligand binding domain of human estrogen receptor can be induced by exposure to 4-hydroxy-tamoxifen. Furthermore, expression of a stable mutant (S33Y) of human beta-catenin is doxycycline inducible. Addition of those drugs separately or in combination allows identification of common or excusive target gene sets.
Project description:To validate the suitability of two commonly used colorectal cancer cell lines, DLD1 and SW480, as model systems to study colorectal carcinogenesis, we treated these cell lines with beta-catenin siRNA and identified beta-catenin target genes using DNA microarrays. The list of identified target genes was compared to previously published beta-catenin target genes found in the PubMed and the GEO databases. Based on the large number of beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study beta-catenin regulated genes and signaling pathways 12 arrays (2 cell lines, 2 treatments, 3 biological replicates)
Project description:Myeloid cell lines (K562 and HEL) were treated overnight with CHIR99021 or a vehicle control (DMSO). CHIR99021 treatment inhibits GSK3B within the destruction complex functioning in Wnt/beta-catenin signalling pathway, thereby preventing beta-catenin degradation and promoting its stabilization. Following the overnight incubation, beta-catenin RIP (RNA immunoprecipitation) was performed in both K562 and HEL cells. RNA samples obtained from beta-catenin RIP in these cells were then sequenced to identify beta-catenin-associated RNAs under CHIR99021 treatment compared to basal conditions (DMSO control).
Project description:Canonical Wnt/B-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no β-catenin nuclear-localization even where cytosolic B-catenin is abundant. Differential propensity for nuclear-localized β-catenin is also observed in cell lines. To investigate the factors mediating the nuclear-localization of B-catenin we carried out a nuclear/cytoplasmic proteomic analysis of the B-catenin interactome in myeloid leukemia cells. From this we identified hundreds of putative novel B-catenin-interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear B-catenin, K562/HEL) versus Wnt-unresponsive cells (low nuclear B-catenin, ML1) suggested the established interactor, LEF1, is a key factor mediating the nuclear-localization of B-catenin in myeloid leukemia. The relative levels of nuclear LEF1 and B-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF1 knockdown inhibited B-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF1 overexpression was able to promote both nuclear-localization and B-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This study is the first to present a B-catenin interactome in hematopoietic cells and reveals LEF1 as a critical regulator of canonical Wnt signaling in myeloid leukemia.
Project description:Deregulation of canonical Wnt/beta-catenin pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 (beta-catenin gene) are highly frequent in colon cancer and cause aberrant stabilization of b-catenin, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of b-catenin by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of beta-catenin in colon cancer cells (GSE53656). Immunoprecipitated samples from human colon cancer SW480 cells with antibodies against beta-catenin and control IgG respectively were used for ChIP-seq experiments.
Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.
Project description:Inositol-Requiring Enzyme (IRE)1 is an evolutionarily conserved sensor protein of the unfolded protein response (UPR). Vertebrates express two distinct paralogues: IRE1α (gene name ERN1) and IRE1β (gene name ERN2). Both proteins have a similar overall structure, with a sensor domain positioned in the endoplasmic reticulum (ER) lumen, and cytoplasmic kinase and endonuclease domains. For IRE1α, it is well-established that a protein folding chaperone called HSPA5 binds to the ER-luminal domain in conditions when the folding load and capacity are balanced. When unfolded proteins accumulate, HSPA5 is recruited to these unfolded proteins to aid in folding, thereby releasing IRE1α. This initiates a chain reaction of dimerization, trans-autophosphorylation and further oligomerization, resulting in an endonuclease-active IRE1α. Activated IRE1α cleaves an intron from the X-box binding protein 1 mRNA in an unusual cytoplasmic splicing reaction, and the resulting frameshift leads to production of a central UPR transcription factor, XBP1S. In contrast to this, IRE1β is far less extensively characterized. It is expressed solely in mucus-producing cells, more specifically goblet cells. Ectopic expression in other cell types such as Hela cells causes rapid cell death, most likely due to unregulated endonuclease activity. Upon aligning the two IRE1 paralogues, it becomes clear that the luminal domain has the lowest homology between the two paralogues, suggesting differences in how activity of either paralogue is tuned in the ER. In this project, we aimed to characterize and compare the IRE1β and IRE1α interactomes in LS174T cells, a cell line that has retained many goblet cell characteristics, to gain more insights into the regulation and downstream activity of IRE1β in a more relevant cellular background.
Project description:Analysis of genes regulated by canonical Wnt signaling in the murine primary Schwann cells. Total RNA from b-catenin fl/fl Schwann cells, after introducing loss-of-function mutations with HTN-cre, or mimicking gain-of-function mutations with Chir98014 or Wnt3a-treatments, was compared to the respective controls.
Project description:In nucleated cells, β-catenin, the key downstream effector of this pathway, is a dual function protein, regulating the coordination of gene transcription and cell–cell adhesion. The specific role of β-catenin in the anucleate platelet however remains elusive. Here, we performed a label-free quantitative proteomic analysis of β-catenin immunoprecipitates from human platelets identifying 9 co-immunoprecipitating proteins. GO biological pathway analysis revealed a significant enrichment of specific functional terms including 'cell adhesion', 'cell junction organization' and ‘adherens junction organization'. Our bioinformatics data suggests that human platelet β-catenin may be involved in facilitating cell adhesion and cell junctions. We found three proteins co-immunoprecipitating with β-catenin under both resting and activated conditions, four proteins under resting condition only and two proteins under activated condition only.
Project description:Here we show that ?-catenin mediates pro-resilient and anxiolytic effects in mice in the nucleus accumbens (NAc), a key brain reward region, an effect that is mediated by ?-catenin signaling in D2-type medium spiny neurons (MSNs) specifically. Conversely, blocking ?-catenin function in NAc promotes susceptibility to chronic stress, and we show evidence of robust suppression of ?-catenin transcriptional activity in the NAc both of depressed humans examined postmortem as well as of mice that display a susceptible phenotype after chronic stress, with a converse upregulation in mice that are stress resilient. Using ChIP-seq, we demonstrate a global, genome-wide enrichment of ?-catenin in the NAc of resilient mice, and specifically identify Dicer1—important in small RNA (e.g., microRNA [miRNA]) biogenesis—as a critical ?-catenin target gene involved in mediating a resilient phenotype. Small RNA-seq after excising ?-catenin from the NAc in the context of chronic stress reveals dynamic ?-catenin-dependent miRNA regulation associated with resilience. Control: 2 samples, Resilient: 2 samples, Susceptible: 2 samples; DNA input: 1 sample.
Project description:During canonical Wnt signalling the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones simultaneously carrying loss-of-function alleles of all four TCF/LEF genes. Exploiting unbiased whole transcriptome sequencing studies, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.