Gene expression profiling in Vav-Etv2 KSL and Granulocyte hematopoietic cells
ABSTRACT: Etv2 transgene was expressed from ROSA26 locus by removing floxed STOP cassette by Vav Cre transgene. KSL or Mac1+/Gr1+ cells were sorted from control or Vav-Etv2 bone marrow and compared for gene expression in duplicate. This study will reveal the effect of Etv2 transgene in adult hematopoietic cells. The effect of Etv2 overexpression in relevant mouse tissue will be important to understand its effect in comparison with in ES cells. Genes aberrantly regulated by Etv2 overexpression will help to understand the caveat when using Etv2 to induce endothelial and hematopoietic cells in vitro. Sample ID #1;KSL;Control #2;KSL;Control #3;KSL;Tg #4;KSL;Tg #5;Gr;Control #6;Gr;Control #7;Gr;Tg #8;Gr;Tg
Project description:Etv2 transgene was expressed from ROSA26 locus by removing floxed STOP cassette by Tie-2 Cre transgene.VE-Cad+/CD45+ cells were sorted from control or tie-2-Etv2 E10.5 embryosYS(yolk sac) and compared for gene expression in duplicate. This study will reveal the effect of Etv2 transgene in E10.5 mouse embryo yolk sac. The effect of Etv2 overexpression in relevant mouse tissue will be important to understand its effect in comparison with in ES cells. Genes aberrantly regulated by Etv2 overexpression will help to understand the caveat when using Etv2 to induce endothelial and hematopoietic cells in vitro. Sample ID YS- ; (VECAD+CD45+;YS;Control) YS+12; (VECAD+CD45+;YS;Tg) YS+22 ; (VECAD+CD45+;YS;Tg)
Project description:Etv2 transgene was expressed from ROSA26 locus by removing floxed STOP cassette by Tie-2 Cre transgene. VE-Cad+/CD45= or VE-Cad+/CD45+ cells were sorted from control or tie-2-Etv2 E11.5 embryos (YS;yolk sac and Emb;embryo proper)and compared for gene expression in duplicate. Sample ID #1 VECAD+CD45-P3;Emb;Control #2 VECAD+CD45-P3;Emb;Tg #3 VECAD+CD45+P4;Emb;Control #4 VECAD+CD45+P4;Emb;Tg #5 VECAD+CD45-P3;YS;Control #6 VECAD+CD45-P3;YS;Tg #7 VECAD+CD45+P4;YS;Control #8 VECAD+CD45+P4;YS;Tg
Project description:Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. Understanding what factors contribute to the variance in tTAV-associated mortality is likely to inform the development of insect control systems. In the present study, the OXI513a tTAV feedback circuit was introduced and verified into D. melanogaster. The tight tetracycline regulation of the system affords a convenient method to conduct a strain-by-strain assessment of the tTAV system and determine the transcriptomic effect, if any, of a positive feedback circuit. Transcriptomic analysis of four independent D. melanogaster tTAV insertion lines, in both adults and larvae, was conducted to examine the transcriptomic influence of the tTAV system. The tTAV lines and non-tTAV control were maintained on TET-on media for 5 generations prior to commencing. Thirty flies, 15 male and 15 female, of the same age, from each of the strains were transferred to either TET-On or TET-Off media. Five days after transfer, adult flies were removed and 10 adult flies, 5 male & 5 female, were frozen at -80°C. Ten larvae from each of these matings were collected at the late second instar stage, the last life stage normally seen prior to lethality, and frozen at -80°C. Three biological replicates were produced for each combination of strain, life-stage, and media. RNA was prepared from frozen samples using Trizol and DNAse was treated using a PureLink RNA Mini Kit (Invitrogen). Microarray experiments employed Agilent Drosophila Gene Expression Microarrays 4x44K and were scanned on an Agilent G2505C scanner (Agilent Technologies). Data collection was divided into two separate experiments. A pilot experiment for strain 102D consisting of two life stages in two conditions each with three biological replicates – a total of 12 samples. The remaining four strains were run as a separate experiment with a total of 48 samples. For each experiment, sample chip position was randomized to avoid genotype- and treatment-specific batch effects.
Project description:To determine the expression of cytochrome P450 genes during normal development Zebrafish embryos were collected at 3, 6, 12, 24, 36, and 48 hours post fertilization. Quadruplicate biological replicates of 100 embryos were hybridized.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations. GMP and MEP were isolated from Runx1+/+-Tg(vav-Cre) and Runx1fl/fl-Tg(vav-Cre) mice as well as Runx1fl/fl-Tg(vav-Cre) XMP, total RNA extracted and sequenced
Project description:We subjected MCF7 cells to starvation with 0.5% charcoal treated serum for 48h and then we added 17-beta estradiol (E2) at final concentration of 10 nM, profiling before and after 60 minutes of treatment the transcriptome and the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation. Comparison of translatome profile changes with corresponding transcriptome profile changes represents a way of studying translational control networks and the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. It is well known that E2 is a strong transcriptional regulator, while its translational control activity is less characterized. To provide a direct experimental evaluation of E2 induced translational regulation, we compared translatome and transcriptome profiles of E2 treated cells. Keywords: polysomal profiling, translatome profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, estradiol stimulation, estrogen receptor. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved MCF7 cells transcriptome in response to E2 stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA and total RNA were isolated from MCF7 cells serum starved and treated with E2. Cells lysates were collected before (t = 0 min) and after (t = 60 min) E2 treatment. All experiments were run in quadruplicates.
Project description:We explored the combinatorial interactions between p53, estrogen receptors and NFkB using the breast adenocarcinoma-derived MCF7 cells. Recently, we have established that p53 can modulate transcription also through non-canonical response elements (REs), consisting of half-sites and ¾ sites. In particular, we identified a half-site p53 response element in the promoter of FLT1/VEGFR-I gene that was required but not sufficient for the p53 responsiveness of the promoter. The transcriptional control required also estrogen receptor (ER) half-sites (EREs) located in close proximity to the p53 RE. Further studies led us to establish that p53-mediated transcription from the FLT1 half site RE is dependent on ER binding at the EREs and strongly influenced by the level of ER proteins, and the specific nature of the p53-inducing stress as well as ER ligand. In another study we found an enrichment of NFkB REs nearby p53 REs in p53 target genes, suggesting an additional mode of functional interactions between these two transcription factors. To identify at a genome scale the transcriptional network that selectively responds to the concomitant activation of p53, ER and/or NFkB, we measured gene expression upon treatment with specific chemotherapeutic agents combined with 17-β estradiol (E2) and/or the inflammatory cytokine TNFα. Firstly we measured using MCF7 cells growing in estrogen-depleted media the transcriptome changes induced by doxorubicin (doxo) and 5-fluorouracil (5FU), two different drugs both able to stabilize and activate the p53 protein. We next obtained the expression profiles in the presence of E2 with or without doxo or 5FU. We also measured transcriptome changes in response to TNFαalone or in combination with doxo and/or E2. All these analyses were conducted using the same batch of MCF7 cells (wild type for p53, ERα, ERβ -weakly-, and NFkB positive) that we had previously used to characterize the cis-mediated transcriptional cooperation between p53 and ER at the FLT1 gene. Keywords: transcription factor, transcriptional networks, p53, ER, NFkB, p53-ER-NFkB crosstalk, doxorubicin, 5-fluorouracil, 17-β estradiol, TNFα, combined genotoxic, estrogenic and inflammatory responses, composite DNA binding site signatures, MCF7. The cooperation at the transcriptional level among p53, ERs and NFkB was analyzed by transcriptome analysis in response to different stimuli (doxo or 5FU, E2, TNFα). To identify mRNA molecules that would be modulated by the coordinated activity of the three TFs, gene expression signals derived from the combinatorial treatment were compared by microarrays analyses to those obtained from each drug when individually administered. Total RNA was isolated from MCF7 cells grown in estrogen-depleted medium and treated so as to stimulate the activity of the three different transcription factors (p53, ERs and NFkB). Cells lysates were collected after 10 hours from the treatments. All experiments were run from triplicate to septuples.
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.