Project description:we have developed a new catalytic tagging system and have used it on the identification of interacting partners of membrane proteins
Project description:we have developed a new catalytic tagging system and have used it on the identification of interacting partners of membrane proteins
Project description:Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP inhibitor (PARPi) resistance using an inducible CRISPR/Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and NHEJ-dependent repair of DNA double-strand breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5’ overhangs through its endonuclease and 3’-5’ exonuclease activities, promotes the repair of DNA lesions including heterochromatic LAD breaks as well as deprotected telomeres, and functions as a downstream effector of DNA-PKcs. These findings underline NUMEN’s role as a key player in DNA repair pathway choice and genome stability maintenance and have implications for ongoing research into the development and treatment of genome instability disorders.
Project description:HeLa cells expressing mCherry-Golgin45, Flag-UBC9-WT/DN and myc-SUMO1 were subjected to Co-IP experiment using RFP-beads (M165-8, MBL life science). Independent triplicates of mCherry-Golgin45 Co-IP experiments were prepared and analyzed by MS independently in a label free format.
Project description:To further stably express PTPRR (WT)-PafA or PTPRR (DA)-PafA in iPUP OVCAR5 cell, we subcloned PTPRR-WT or DA, respectively, into the PafA-IRES-EGFP plasmid. Each plasmid was packed into a lentivirus and then transduced into iPUP OVACR5 cells for 48 h. GFP-positive cells were sorted by flow cytometry. The expression of PTPRR (WT)-PafA and PTPRR (DA)-PafA was confirmed by western blotting analysis. PTPRR (WT)-PafA or PTPRR (DA)-PafA expressed iPUP OVCAR5 cells were grown to a cell density of about 75% on 10 cm dishes. We followed the protocol established previous. To prepare PUP-IT samples for mass spectrometry analysis, including doxycycline induction, biotin labeling, cell lysis, streptavidin magnetic beads pull-down, trypsin digestion, and peptide cleaning.
Project description:Accumulating evidence highlights the role of long non-coding RNAs (lncRNA) in cellular homeostasis, and their dysregulation in disease settings. Most lncRNAs function by interacting with proteins or protein complexes. While several orthogonal methods have been developed to identify these proteins, each method has its inherent strengths and limitations. Here, we combine two RNA-centric methods ChIRP-MS and RNA-BioID to obtain a comprehensive list of proteins that interact with the well-known lncRNA HOTAIR. Overexpression of HOTAIR has been associated with a metastasis-promoting phenotype in various cancers. Although HOTAIR is known to bind with PRC2 and LSD1 protein complexes, an unbiased and comprehensive method to map its interactome has not yet been performed. Both ChIRP-MS and RNA-BioID data sets show an association of HOTAIR with mitoribosomes, suggesting HOTAIR has functions independent of its (post-)transcriptional mode-of-action.
Project description:Little is known about the epigenomics of liposarcoma (LPS). Here, we profiled the global expression of 9 epigenetic marks in well differentiated (WD) and dedifferentiated (DD) LPS from 151 patients and found increased H3K9me3 among DDLPS tumors. We performed ChIP-seq and gene expression profiling of patient derived cell lines to discover functionally significant regions of differential H3K9me3 enrichment between WDLPS and DDLPS associated with concomitant gene expression changes. We performed genome-wide transcriptional profiling of H3K9me3 in dedifferentiated liposarcoma DDLPS and well differentiated liposarcoma WDLPS cell lines.
Project description:The active form of the SREBP2 transcription factor was specifically overexpressed in the intesitne. This was achieved by generating transgenic mouse model (designated as ISR2) in which the transgene represents the N-terminal active SREBP2 transcription factor driven by the villin promoter. In this project, we investigated the effects of overactivation of SREBP2 on gene expression in mouse jejunum Gene expression profile in the jejunum of ISR2 was compared to gene expression profile in the jejunum of their wild type littermates.
Project description:The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Igκ and Tcrα J gene segments and Igh and Tcrβ J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding is restricted to regions containing recombination signal sequences, RAG2 binds extremely broadly in a pattern that mirrors that of trimethylated lysine 4 of histone 3. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination. RAG2 binding was analyzed in wild type, RAG2-/-β, and D708A-RAG1-/-β thymocytes. Histone modification H3K4me3 was analyzed in D708A-RAG1-/-β and wild type thymocytes.
Project description:Mitochondrial function is modulated by its functional interaction with the endoplasmic reticulum. Recent research indicates that these contacts are disrupted in familial models of amyotrophic lateral sclerosis. We report here this impairment in the crosstalk between mitochondria and the endoplasmic reticulum impedes the use of glucose-derived pyruvate as mitochondrial fuel, causing a shift to fatty acids to sustain energy production. Over time, this deficiency alters mitochondrial electron flow and the active/dormant status of complex I in spinal cord tissues, but not in the brain. These findings suggest MAM plays a crucial role in regulating cellular glucose metabolism and that its dysfunction may underlie the bioenergetic deficits observed in ALS.