Project description:The Mdm2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that Mdm2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, Mdm2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the Mdm2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. Mdm2 physically associated with EZH2 on chromatin, enhancing the trimethylation of Histone 3 at lysine 27 and the ubiquitination of Histone 2A at lysine 119 (H2AK119) at its target genes. Removing Mdm2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, Mdm2 supports the Polycomb-mediated repression of lineage specific genes independent of p53. microarray analysis in HCT116 p53-/-cells
Project description:The Mdm2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that Mdm2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, Mdm2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the Mdm2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. Mdm2 physically associated with EZH2 on chromatin, enhancing the trimethylation of Histone 3 at lysine 27 and the ubiquitination of Histone 2A at lysine 119 (H2AK119) at its target genes. Removing Mdm2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, Mdm2 supports the Polycomb-mediated repression of lineage specific genes independent of p53. microarray analysis in osteoblasts differentiated from human mesenchymal stem cells after siRNA kd
Project description:The Mdm2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that Mdm2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, Mdm2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the Mdm2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. Mdm2 physically associated with EZH2 on chromatin, enhancing the trimethylation of Histone 3 at lysine 27 and the ubiquitination of Histone 2A at lysine 119 (H2AK119) at its target genes. Removing Mdm2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, Mdm2 supports the Polycomb-mediated repression of lineage specific genes independent of p53. H3K27me3 and H2Ak119ub1 ChIP-Seq
Project description:Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Four histopathological patterns of MS have been described. Pattern II is characterized by antibody and complement deposition. MS is considered a prototypic T cell-mediated disease, but until now the study of pathogenic T cells has encountered major challenges, most importantly the limited access of brain-infiltrating T cells. Here, we used next generation sequencing to identify clonally expanded T cells in demyelinating pattern II brain autopsy lesions and subsequently isolated these as T cell clones from autologous cerebrospinal fluid. The functional characterization shows that T cells releasing Th2 cytokines and able to provide B cell help dominate the T cell infiltrate in pattern II brain lesions. Our data provide the first functional evidence for a role of Th2/Tc2 cells in pattern II MS. Two stimulated CD4+ Th2 brain infiltrating T cell clones compared with stimulated circulaiting memory CD4+ T cells and two stimulated CD8+ T cell clones (one Tc1 and one Tc2) compared with each other.
Project description:High-density lipoproteins (HDLs) protect pancreatic β cells against apoptosis. This property might be related to the increased risk to develop diabetes in patients with low HDL blood levels. However, the mechanisms by which HDLs protect β cells are poorly characterized. Here we use a transcriptomic approach to identify genes differentially modulated by HDLs in β cells subjected to apoptotic stimuli. Experiment Overall Design: Tested conditions on beta-TC3 cells for 6h (in triplicates for each condition; GSK3 and GSK4 indicate two independent HDL preparations): Experiment Overall Design: Complete Medium + Vehicle [V] Experiment Overall Design: Complete Medium + HDL (prep. HDL GSK3 or HDL GSK4) [HDL] Experiment Overall Design: Starvation medium + Vehicle [V.S] Experiment Overall Design: Starvation medium + HDL (prep. HDL GSK3 or HDL GSK4) [HDL.S]
Project description:Comprehensive profiling of the cell surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here, we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step that we called plasma membrane-BioID. The lipid modified BirA* enzyme (MyrPalm BirA*) was targeted to the plasma membrane where it effectively biotinylates plasma membrane proteins. Biotinylated proteins were then affinity purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and plasma membrane-BioID is ideal to overcome the inherent limitations of identification of integral membrane proteins and yields highly pure plasma components for downstream proteomic analysis.
Project description:miR-101 is a tumor suppressive microRNA. Besides EZH2, a well known EZH2 target, miR-101 should have other targets, which might also contribute to its tumor suppressive function. Thus, we performed array hybridization to determine changes in gene expression upon miR-101 overexpression in U2OS cells. U2OS were transfected with synthetic miRNA negative control (NC) or miR-101 for 48hrs, and then harvested for RNA preparation. Three independent experiments were performed. One NC sample were repeated twice for the microarray.
Project description:Regulation of protein output at the level of translation allows for a swift response to dynamic changes in the requirements of the cell. This is achieved by a complex set of biochemical processes that regulate protein synthesis and mRNA stability. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Bioreplicate one for this paper is available at the Proteome xchange identifier: PXD014764
Project description:Thyroid carcinoma (TC) is generally associated with good prognosis, nevertheless no effective treatments are available for aggressive forms not cured by current therapies. We previously identified the coatomer protein complex zeta 1 (COPZ1), as a new putative therapeutic target for TC, since its depletion impairs the viability of tumor cells, leads to abortive autophagy, ER stress, unfolded protein response and apoptosis, and reduces the tumor growth of TC xenograft models. In this study, by combining genomic, proteomic and functional approaches, we provided evidence that COPZ1 silencing stimulates a type I IFN-mediated viral mimicry response, boosts the production of several inflammatory molecules and finally induces immunogenic cell death, which, in turn, promotes dendritic cell maturation and subsequent activation of T cells. Collectively, our findings support the notion that COPZ1 targeting can be exploited as a new strategy to kill cancer cells with the subsequent involvement of an anti-tumor immune response.