Project description:The analysis of differential interactome of Pol II pS5 in PHF3 WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (4H8) antibody coupled to Protein G beads.
Project description:The analysis of differential interactome of Pol II pS5 in DIDO WT, KO and ΔSPOC HEK293T cells using Pol II pS5 (3E8) antibody coupled to Protein G beads (no crosslinking).
Project description:DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.
Project description:In contrast to our extensive knowledge on covalent SUMO target proteins, we are limited in our understanding of proteins that bind SUMO family members in a non-covalent manner. We have identified interactors of different SUMO isoforms: monomeric SUMO1, monomeric SUMO2 or linear trimeric SUMO2 chains, using a mass spectrometry-based proteomics approach. We identified 382 proteins that bind to different SUMO isoforms mainly in a preferential manner. Interestingly, XRCC4 was the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2 as well as the only protein in our screen that belongs to the Non-Homologous End Joining (NHEJ) DNA double-strand break repair pathway. A functional SIM in XRCC4 regulated its recruitment to local sites of DNA damage and its phosphorylation in S320 by DNA-PKcs. Combined, our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provides a resource of SUMO isoform interactors.
Project description:Septin proteins interact intermolecularly to form hetero-oligomeric complexes that further assemble into higher-order filaments. To investigate whether the interactions among septin proteins are affected by RID, we conducted affinity purification-mass spectrometry (AP-MS) experiments. In this regard, EGFP-tagged SEPT6 or its quadruple mutant SEPT6-4KR was co-expressed with RID-WT or RID-CA in cells and immunoprecipitated by anti-EGFP beads. The resulting co-immunoprecipitates were analyzed and compared with the vector control by label-free quantitative (LFQ) proteomics to identify and quantify septin proteins associated with SEPT6. The MS analysis showed that SEPT6 were indeed co-affinity-purified with all endogenous septin proteins that were identified, such as SEPT2, SEPT3, SEPT5, SEPT6, SEPT7, SEPT8, SEPT9, SEPT10, and SEPT11, in the absence of RID activity. More importantly, the interactions between SEPT6 and these septins were not affected by RID-WT expression. Interestingly, the SEPT6-4KR mutant could still interact and likely form heteromeric complexes with other fatty-acylated septin proteins (e.g., SEPT2, SEPT3, SEPT5, SEPT6, SEPT7, SEPT8, SEPT9, SEPT10 and SEPT11) in the presence of RID activity.
Project description:H3K27ac and H4K5ac ChIPseq in WT and Hdac3-/- mESCs in order to determine the role of Hdac3 on the activity of cis-regulatory elements during differentiation.<br>This experiment is part of the study: "Mechanistic framework of lineage restriction in embryonic stem cells" by Olivieri et al.
Project description:To better delineate how ROCK1 controls the biochemical state of activated B cells, we next conducted a phospho-proteomic analysis. In vitro stimulated WT and CD23-Rock1 B cells were harvested, subjected to pS/pT TMT and total proteome TMT, and results analyzed by Proteome Discoverer.
Project description:Quantitative chemical proteomics was performed to profile the target proteins of ITCs.For the forward SILAC experiment, heavy-labeled cells were treated with the ITC probe (20 μM) for 30 min and light-labeled cells were pre-treated with the natural ITC (60 μM) for 30 min, followed by the ITC probe (20 μM) for another 30 min. For the reverse SILAC experiment, light-labeled cells were treated with the ITC probe (20 μM) for 30 min and heavy-labeled cells were treated with the natural ITC (60 μM) for 30 min, followed by the ITC probe (20 μM) for another 30 min.
Project description:FLAG-hPHF3 was transiently expressed in human HEK293 cells, followed by anti-FLAG immunoprecipitation, on-bead digest and NanoLC-MS analysis.
Project description:Gastrulation is a critical stage of embryonic development during which the three germ layers are established. Deciphering the molecular mechanisms underlying this proces from a protein perspective remains a significant challenge. To address this, we employed a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho)protein expression during differentiation of ESCs towards gastruloids – an in vitro model of gastrulation-stage embryogenesis. Our findings revealed that many proteins exhibited temporal expression with unique expression profiles corresponding to the three germ layers. Additionally, we profiled enhancer interaction landscapes in ESCs and gastruloids using p300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron based perturbations combined with scRNA-seq revealed a critical role for Zeb2 in regulating mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.