Project description:Although ubiquitination of DX2, novel oncogene, is significant on its protein stability, but DX2 ubiquitination sites are not unveiled. In this study, we identified ubiquitination sites of DX2 upon treatment with DX2 protein inhibitor, SDL01, via LC-MSMS technique. Lysine 90 of DX2 was revealed as a ubiquitination site and was validated as a real ubiquitination site via molecular biology
Project description:To characterize the mechanism of DMC1 ubiquitination as well as further explore the impact of DMC1’s ubiquitination on meiosis, we attempted to identify the potential DMC1 ubiquitination sites. The in vitro ubiquitination assay using recombinant DMC1 heterologously expressed and purified from E. coli as substrate was conducted. Then mass spectrometry assay was performed to identify the ubiquitination sites of DMC1. Five DMC1 lysine residues (K45, K70, K101, K162, and K290) were identified as potential ubiquitination sites.
Project description:Identification of ubiquitination sites on my protein of interest (TRIM52) through NiNTA pull-down of TRIM52 modified by his-ubiquitin.
Project description:Proteolysis Targeting Chimeras (PROTACs) hijack the ubiquitin proteasome system to ubiquitinate target proteins, targeting them for degradation by the proteasome. An understudied process that is essential to the PROTAC mechanism of action is ubiquitination of the target protein and ubiquitin (Ub) chain elongation. To elucidate the mechanism of PROTAC-induced ubiquitination of BRD4(BD2) ubiquitination sites on Brd4BD2 were mapped by mass spectrometry following an in vitro ubiquitination assay containing neddylated-CRL2VHL, Brd4BD2, MZ1, Uba1 and either UBE2R1 or UBE2D2. Time points were taken at 0 minutes, 10 minutes, 0.5 hours, 1 hour and 3 hours. Proteins were separated by SDS-page analysis. To visualize proteins the gel was stained with Coomassie blue and gel slices containing ubiquitin modified Brd4BD2 across timepoints and increasing molecular weight were excised from the gel and were analyzed by mass spectrometry. To identify sites of ubiquitination a search for GG-K peptides was performed in MaxQuant. Based on existing structures the modified lysine residues were all situated on the same face of BD2.
Project description:To identify the ubiquitination sites on Merlin, Flag-tagged wild-type Merlin or the S518D mutant were stably transduced into LN229 cells. These cells were treated with thapsigargin for 15 minutes and subjected to immunoprecipitation and followed by Mass Spectrometry.
Project description:We present Smart-Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2) for the identification and quantification of transcription start sites. Smar2C2 allows for the identification of upwards of 70 million unique transcription start sites from a single sample with as little as 40 pg of RNA input.
Project description:The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23-/- (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-contolled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation. To examine the enrichment of H2B ubiquitination, Pol II, H3K4me3, H3K79me3 in WT and KO MED23 MEF cells, we performed H2Bub ChIP-seq, Pol II ChIP-seq, H3K4me3 ChIP-seq and H3K79me3 ChIP-seq assays. 10 high-throughput sequencing data were deposited and WT, KO input data were controls for peak calling.