Project description:We have expressed mouse Snai1, mutant Snai1 or empty vector in the HPC7 cell line and performed ChIP sequencing analysis of Snai1, H3K4me1 and H3K4me2
Project description:Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we demonstrate that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein halflife, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKGmediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.
Project description:The protein Dicer is required for microRNA (miRNA) biogenesis, and therefore Dicer-deficient cells lack all mature, functional miRNAs. Here we investigated the binding of the PRC2 component Ezh2 in wildtype and Dicer-deficient mouse embryonic stem cells genomewide using ChIP-sequencing analysis. Examination of Ezh2 binding in mouse ES cells either proficient (wildtype) or deficient (KO) of the protein Dicer
Project description:To better understand the OLIG2 binding site in the whole-genome of mouse neural stem cells, ChIP was performed using 107 mouse neural stem cells. The ChIP-Seq library was constructed by using DNA SMART ChIP-Seq Kit according to the manufacturer's instructions (Clontech) and was sequenced on the Illumina HiSeq 2000 Sequencer. OLIG2 Chromatin immunoprecipitation sequencing (ChIP-Seq) in mouse neural stem cell.
Project description:Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful tool to dissect global epigenetic landscapes of cells. However, this method usually consumes millions of cells. Here we develop a robust technique for performing ChIP-Seq using as low as 1,000 cells. This method combines a semi-automatic nanoliter ChIP reaction with a carrier-based sequencing library preparation strategy without pre-amplifying the ChIP product. We used this method to investigate the pattern of trimethylation of histone 3 lysine 4 (H3K4me3) of mouse post-implantation epiblast cells at embryonic day 6.5 (E6.5) and showed that it is more similar to that of mouse Epi-Stem cells (mEpiSCs) than that of mouse Embryonic Stem cells (mESCs). Together with the high similarity between the transcriptomes of EpiSCs and E6.5 epiblast cells, this suggests that mEpiSCs is a reliable in vitro model for post-implantation epiblast cells in vivo. Use 1 million mEpiSCs and 1million mESCs as the positive control, 1000 mEpiSC and 1000 mESCs samples are used to validate the protocol. 1000 mEpiblasts have two biological replicates
