Gene expression analysis after selective expression of tissue factor isoforms
ABSTRACT: Analysis of TF isoform-induced breast cancer cell transformation at gene expression level. The hypothesis tested in the present study was that expression of TF isoforms upregulate genes involved in proliferation and transformation, while downregulating genes involved in cell cycle arrest and apoptosis. Results provide important information on the cellular response to TF expression with respect to pro-oncogenic programs . Total RNA obtained from an MCF-7-based cell model (2A3-3) expressing full length tissue factor (2A3-3-flTF) or alternatvively spliced tissue factor (2A3-3-asTF) compared to cells expressing an empty vector control (2A3-3-pcDNA).
Project description:Transcriptional profiling of Human Embryonic Kidney(293T) Cells comparing control untreated 293T cells with 293T cells transfected with A) pcDNA 3.0(-) vector[Invitrogen] (Mock) and B) Expression vector pcDNA 3.0(-) containing cloned Influenza virus H5N1and H11N1-NS1 (Non-Structural1) gene. Overall design: Two-condition experiment, 293T vs. TF-293T cells. Biological replicates: 3 control, 3 transfected, independently grown and harvested for each set of experiment.Total 4 groups 1.Control untransfected (293T) cells 2. Mock 293T cells transfected with pcDNA3.0(-) expression 3. 293T cells transfected with NS1 gene of H5N1 virus cloned in pcDNA 3.0(-) vector 4.293T cells transfected with NS1 gene of H11N1 virus cloned in pcDNA 3.0(-) vector.
Project description:It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression. For expression profiling of cells in NME2-induced conditions, A549 cells were transfected with pcDNA-NME2-MYC or pcDNA-MYC (control). RNA was isolated from the cells 48h after transfection using the trizol method (Sigma) as per manufacturer’s protocol. Total RNA was processed to hybridize to Illumina Human HT-12 v4 Expression BeadChip as per manufacturer’s instructions. Three biological replicates were averaged and data was analyzed using BeadStudio (P <0.05 of fold change).
Project description:Analysis of coagulation factor FVII-induced breast cancer cell transformation at gene expression level. The hypothesis tested in the present study was that expression of FVII upregulates genes involved in epithelial-to-mesenchymal transition and transformation. Results provide important information on the cellular response to tumor FVII expression with respect to pro-oncogenic programs . Overall design: Total RNA obtained from an MDA-MB-231-based cell line (MDA-FRT; an MDA-MB-231 cell line containing a flp-in FRT site) containing human FVII cDNA (2A3-3-flTF) compared to cells expressing an empty vector control (pcDNA).
Project description:The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for its transforming activity. Here we use hematopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300, to examine the requirement for this interaction in myeloid transformation and leukemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300, we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus. These data highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML and suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. Finally we have begun to explore the molecular basis of the these observations by gene expression profiling; this highlighted several genes previously implicated in myeloid leukemogenesis as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. Total RNA was obtained from FACS sorted GFP+;c-Kit+ primary bone marrow cells from WT and Booreana mouse strains which had been cultured for 48 hours post-transduction with Control or AML1-ETO9a retroviruses. RNA was extracted from each of 4 samples per group and used to probe Illumina mouse Beadchips array.
Project description:Novel target genes for the two transcription factor isoforms Pax6 and Pax6(5a) were identified by generating mouse embryonic fibroblast cell lines stably expressing either Pax6 or Pax6(5a), and comparing their gene expression pattern with the original mouse embryonic fibroblast cell line. Flp-In-3T3 cell lines were generated according to the manufacturers instruction (Invitrogen), using the mouse Pax6 and Pax6(5a) cDNA as insert. Correct inserts were verified by PCR, sequencing and Westernblot.
Project description:We used the ERMYB cell line (Hogg et al., Oncogene 15, p2885-2898, 1997) as our model system to identify genes regulated by Myb using whole genome microarray expression profiling. ERMYB is a myeloid progenitor cell line derived by transformation of primary cells by an activated form of Myb (CT3-Myb) fused to the ligand binding domain of ERα. In these cells, activation of the ER-Myb fusion protein by estrogen is required to maintain a proliferative progenitor-like phenotype. The cells undergo monocytic differentiation when Myb is inactivated by withdrawal of β-estradiol (β-E2). However, re-induction of Myb within 24 hours can almost fully reverse the differentiation process. This feature enabled us to employ a novel kinetic expression profiling strategy, in that we withdrew β-E2 to inactivate Myb for 24 h, then re-added it for another 6 h and monitored the gene expression across this time course. We reasoned that this strategy may be more likely to identify true Myb target genes than conventional approaches in which differential expression is examined only at the endpoint of induction. To exclude genes that might be responsive to ERα and not Myb we used the parent CT3-Myb cell line and measured the expression changes 6hrs after β-E2 withdraw and excluded those few gene that were differentially expressed. Triplicate total RNA samples from ERMYB cells were collected at 0, 6, and 24 hr after β-E2 withdrawal and 6 hr after β-E2 re-addition. Samples were analyzed by Illumina Mouse Whole Genome arrays (WG6). Probe intensities were variance stabilised and normalized by the Lumi package (Du et al., 2008). Differentially expressed genes were identified by the LIMMA package (Smyth, 2004) and then grouped into different classes according to their kinetic profiles.
Project description:Analysis of transcriptional changes during Myc or PI3K induced oncogenic transformation in RWPE1 cells (benign prostate epithelial cell line). The aim of the present study was to identify key epigenetic gene silencing events that occur during the oncogenic transformation events, hence emphasis was placed on downregulated genes. Results provide important information on which are the tumor suppressive pathways or genes that will be epigenetically silenced by during Myc or PI3K induced oncogenic transformation. Total RNA obtained from oncogenic transformed RWPE1 cells which were either overexpressing Myc or constitutive active mutant of PI3K (E545K) as compared to RWPE1 cells expressing the empty vector control PMN.
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.
Project description:3 groups of diet (1, 2, and 3) at two time points (Pre- and Post-diet). 3 diets differed in the amount and type of protein consumed. Diet 1: adequate protein, low dairy (APLD: 15% daily calories from protein, 0-1 servings of dairy); diet 2: adequate protein, moderate dairy (APMD: 15% daily calories from protein, 3-4 servings/d); diet 3: high protein, high dairy (HPHD: 30% daily calories from protein, 6-7 servings of dairy). Randomized controlled trial, repeated measures design, all groups exercised, main difference was nutrition.
Project description:The goal of this study is to investigate the transcriptional network regulated by DIAPH3 gene (mDia2) using the conditional hematopoietic specific knockout mouse model, especially in the cell proliferation, cell survive (including oxidative stress), cell adhesion & migration, cell cycle and hematopoiesis pathway-related genes. Two groups of mice (WT vs KO) were sacrificed to purify erythroblast (Ter119+) cells from total bone marrow for RNA preparation. Each group contains three mice, the WT group includes 917,1125,1136 and 907,914,1122 belong to the conditional KO group. There is biological triplication in each group.