Project description:The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling. Consistent with this, overexpression of FAM83F activates Wnt signalling, whilst ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1α at the plasma membrane. This interaction with CK1α is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1α fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of GSK-3β, because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant of FAM83F in cells through CRISPR/Cas9 genome editing redirects the FAM83F-CK1α complex away from the plasma membrane and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.
Project description:Since the early 1980s remarkable progress has been made in understanding the role of the HER2 locus in carcinogenesis, but many details of its regulatory network are still elusive. We recently reported the finding of 367 new human microRNA (miRNA) genes of which one, mir-4728, is encoded in an intron of the HER2 gene. Here, we confirm that the HER2 oncogene is a bi-functional locus encoding the membrane receptor and a functional miRNA gene. We further show that miR-4728-3p has alternative functionalities depending on the region used for interaction with its target; the canonical seed between nucleotides 2-8 or a novel, more internal seed shifted to nucleotides 6-12. Analysis of public data shows that this internal seed region, although rare compared to the far more abundant canonical 2-8 seed interaction, can also direct targeted down-regulation by other miRNAs. Through the internal seed, miR-4728-3p regulates expression of estrogen receptor alpha, an interaction that would have remained undetected if classic rules for miRNA-target interaction had been applied. In summary, we present here an alternative mode of miRNA regulation and demonstrate this dual function of the HER2 locus, linking the two major biomarkers in breast cancer. 6 samples treated with hsa-miR-4728-3p mimic and 6 samples treated with non-targeting control 4 samples treated with hsa-miR-1-3p mimic and 4 samples treated with non-targeting control
Project description:Neurons and oligodendrocytes communicate to regulate oligodendrocyte development and ensure appropriate axonal myelination. Here, we show that Glycerophosphodiester phosphodiesterase 2 (GDE2) encodes a neuronal pathway that promotes oligodendrocyte maturation through the release of soluble neuronally-derived factors. Mice lacking global or neuronal GDE2 expression have reduced mature oligodendrocytes and myelin proteins but retain normal numbers of oligodendrocyte precursor cells (OPCs). WT OPCs cultured in conditioned medium (CM) from Gde2 null (Gde2KO) neurons exhibit delayed maturation, recapitulating in vivo phenotypes. Gde2KO neurons show robust reduction in canonical Wnt signaling and genetic activation of Wnt signaling in Gde2KO neurons rescues in vivo and in vitro oligodendrocyte maturation. Phosphacan, a known stimulant of OL maturation, is reduced in CM from Gde2KOneurons but is restored when Wnt signaling is activated. These studies identify GDE2 control of Wnt signaling as a neuronal pathway that signals to oligodendroglia to promote oligodendrocyte maturation.
Project description:Up-regulation of canonical Wnt signaling has been implicated in liver fibrosis and cancer. To determine canonical Wnt target genes potentially implicated in these diseases, we performed microarray analysis of culture-activated rat hepatic stellate cells (HSCs) treated with canonical Wnt inhibitors, DKK-1, ICG-001, or FJ9.
Project description:A20 is known key regulator of NF-κB activity and inflammatory response, but its role in the control of cell death receptor signaling is not completely understood. Although A20 is widely accepted anti-apoptotic protein, we demonstrate that elevated expression of A20 in both, human and murine keratinocytes results in sensitisation to TNF-induced cell death. We prove that the Ripoptosome formation in A20 overexpressing cells is prerequisite for the TNF-induced cell death execution. We demonstrate that both canonical and non-canonical NF-κB signaling pathways are regulated upon increase of A20 expression. A20 dependent alteration of cIAPs and TRAF1 expressions are involved in the multiple level control of cell death in keratinocytes with elevated A20 expression.
Project description:A20 is known key regulator of NF-κB activity and inflammatory response, but its role in the control of cell death receptor signaling is not completely understood. Although A20 is widely accepted anti-apoptotic protein, we demonstrate that elevated expression of A20 in both, human and murine keratinocytes results in sensitisation to TNF-induced cell death. We prove that the Ripoptosome formation in A20 overexpressing cells is prerequisite for the TNF-induced cell death execution. We demonstrate that both canonical and non-canonical NF-κB signaling pathways are regulated upon increase of A20 expression. A20 dependent alteration of cIAPs and TRAF1 expressions are involved in the multiple level control of cell death in keratinocytes with elevated A20 expression.
Project description:The histone 3 lysine 9 (H3K9)-specific methyltransferase (KMT) Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on Setdb1 role(s) in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for muscle adult stem cells expansion following activation and in vitro studies on skeletal myoblasts confirmed that Setdb1 suppresses terminal myoblast differentiation. We used genome-wide analyses to identify Setdb1 direct target genes in myoblasts and observed a release of Setdb1 from the promoter of selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. We demonstrated that both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Taken together, our findings uncover a functional link between Setdb1 and canonical Wnt signalling in skeletal muscle cells, which affects the expression of a subset of Setdb1 target genes. We revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions. ChIP-seq of Setdb1 and H3K9me3 in Myoblast cells (C2C12)