Transcription profiling by high throughput sequencing of mouse embryonic stem cells following Trim24 and/or p53 knockdown
ABSTRACT: Differentially expressed genes were determined by single-end sequencing (stranded protocol) following Trim24 and/or p53 knock down in the mouse ES cells grown in 2i media. Triplicates were generated for treatment and control samples but for p53 knock down (duplicate).
Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem ce ...[more]
Project description:ChIP followed by next generation sequencing against Trim24 was performed in mouse embryonic stem (ES) grown either in 2i+LIF (2iL) or serum media. Two biological replicates for each condition were sequenced using Illumina HiSeq. Two input controls (no IP) were also generated for each condition.
Project description:In mouse embryonic stem cells (mES cells), ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes as well as Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation. mouse ES cell mRNA profiles of control, Ring1B knock down, and Dzip3 knock down, in duplicate, using Illumina MiSeq.
Project description:Specific surface marker for NKX2-1+ VAFECs may be helpful for isolating a homogeneous population of alveolar epithelial progenitor cells and distinguishing the differentiation from a thyroid lineage to a lung lineage. In order to identify specific markers of VAFECs, a microarray analysis was performed to compare the global gene expression patterns between AFECs and VAFECs in 201B7 hiPSCs. We hypothesized that NKX2-1+ cells could be purified by sorting CPM+ VAFECs. After dissociating VAFECs cells on day 14 with Accutase, FACS was performed using anti-EPCAM and anti-CPM antibodies. EPCAM+CPM+ and EPCAM+CPM- cells were then sorted, and the global gene expression patterns of these two populations were examined using a microarray analysis. In addition, MACS was performed to obtain CPM+ cells for comparison. We extracted total RNA from hiPSCs-derived AFECs, VAFECs, EPCAM+CPM+ and EPCAM+CPM- VAFECs and CPM+ VAFECs and hybridized them to Affymetrix microarrays.
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
Project description:The role of p53 in assuring longevity through prevention of cancer is well established, but how it specifically regulates aging is still controversial. Our assumption is that distinct p53-pathways regulate tumor suppression and aging and that p66Shc is one of the master regulators of the p53 aging function. p66Shc longevity determinant protein acts as a downstream target of p53 and it is indispensable for the ability of activated p53 to induce elevation of intracellular oxidants and apoptosis. We used microarray to gain insight into the mechanism through which p66Shc could regulate p53 oxidative stress dependent activity. Total RNA was extracted from Primary murine embryonic fibroblasts (MEFs) were isolated from 13.5-day-old embryos according to standard procedures from WT, p53KO and p66ShcKO mice. Treatment: Early passage (p.2) MEFs were washed twice with PBS1X solution, and then incubated in DMEM media containing 400 μM of H2O2 or 0.25μg/ml of Doxorubicin (SIGMA) at 37°C for four hours.
Project description:We investigated the genomewide binding pattern of prevalent p53 gain-of-function (GOF) mutants by ChIP-seq, in a panel of breast cancer cell lines. We assessed the genomewide changes of H3K4me3 upon GOF p53 knockdown in MDA-MB-468 breast cancer cells bearing the p53 R273H mutation. This study uses ChIP-seq of H3K4me3 and histone H3 in wild-type or p53 R172H knock-in MEFs. Additionally, this study examines the transcriptome of wild-type or p53 R172H knock-in MEFs using polyA+ RNA-seq.
Project description:In the present study, we used a high-throughput small RNA deep sequencing followed by a systematic computational analysis to identify genome wide mutant p53R273H regulated miRNAs in both DNA damage dependent and independent context. Several miRNA-mRNA regulatory networks have been predicted that might contribute to mutant p53 GOF properties. Differentially regulated miRNA signature profile has been validated in the lung cancer patients harboring wildtype and mutant p53. We identified specific miRNA signatures for lymph node metastasis associated with p53 mutation in lung adenocarcinoma and also predicted the possible contribution of two mutant p53 regulated miRNAs in EMT process. Furthermore, this study identified a hitherto unknown miRNA in human which might act as one of the crucial downstream targets of GOF mutant p53 to confer oncogenic properties. Determination of mutant p53R273H regulated microRNAs H1299 cells.
Project description:We sequence mRNA from single mESCs from three culture conditions: serum + LIF, 2i + LIF and alternative 2i + LIF. We extensively analysed population and single cell gene expression to identify differences and similarities between conditions. This is a subset of data from ArrayExpress accession E-ERAD-186 (ENA: ERP003293)
Project description:Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ Xist∆A/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v22.214.171.124, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Total RNA was extracted and purified from each cell type and their transcriptomes analysed by RNA-Seq.