Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well-conserved across variants and is critical for suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus-host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we co-immunoprecipitated WT Mac1 and Mac1 N40D from HEK293T and A549 cell lines and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding.
Project description:We report a significant differential expression of proteins, all of which are associated with a viral infection response typified by Interferon type I/II. Notably the upregulated proteins may affect several vital cellular processes, including cell cycle control, translation and post-translational modification of proteins.
Project description:This study used the BioID proximity assay to establish the BACE1 interactome in healthy neuronal cells and identified interactions involved in BACE1 trafficking, post-translational modification and substrates.
Project description:SFRP1 functions as an important inhibitor of the Wnt pathway and is a known tumor suppressor gene, which is epigenetically silenced in a variety of tumors. Nevertheless, too little is known about signaling pathways modulated by forced SFRP1 re-expression in dependency of the molecular breast cancer subtypes. In this study, we performed a systematic expression analysis of stably transfected human breast cancer cells to determine those molecules and biochemical pathways affected after forced SFRP1 re-expression.
Project description:We report that full length TET1 (TET1-FL) overexpression fails to induce global DNA demethylation in HEK293T cells. The preferential binding of TET1-FL to hypomethylated CpG islands (CGIs) through its CXXC domain leads to its inhibited 5-hydroxymethylcytosine (5hmC) production as methylation level increases. TET1-FL-induced 5hmC accumulates at CGI edges, while TET1 knockdown induces methylation spreading from methylated edges into hypomethylated CGIs. However, TET1 can regulate gene transcription independent of its dioxygenase catalytic function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically maintains the DNA hypomethylation state of CGIs in adult cells. Genome-wdie profiling of gene expression in HEK293T cells following overexpression of wild type or catalytically mutant TET1-FL or TET1-CD
Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs
Project description:RNA from four independent cultures from each sh Kdm5c #1, sh Kdm5c #2 and non-targeting shRNA polyclonal cell lines were hybridized in dye-swap against a common reference of RNA from IB10 ES cells
Project description:We performed RNA-seq experiments on three biological replicates of HeLa cells depleted of MATR3, PTBP1/2, controls, or combined depletion of MATR3/PTBP1/2. Cells were fractionated into cytoplasmic and nuclear RNA. Library preparation was done with the TruSeq stranded RNAseq library kit (Illumina) according to manufacturer’s recommendations; RNA was depleted of rRNA using the RiboZero kit (Epicentre). All libraries were sequenced on Illumina HiSeq2 machines in a single-end manner with a read length of 100 nt.
Project description:The transcriptomic dose-dependent response of rat immortalized thyrocytes to two thyroid disrupting chemicals was investigated by RNA-seq experiment. PCCl3 cells were exposed for one week to different doses of ethylenethiourea (ETU) and chlorpyrifos (CPF) and to their combinations.