Project description:We hypothesized that ETV5 may be a mediator of the oncogenic effects of mutant FGFR3 in bladder cancer cells ETV5 was silenced by shRNA in the bladder cancer cell line 97-7 to investigate effect on phenotype. To identify downstream gene targets of ETV5 we compared gene expression profiles in silenced and control cells.
Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro. Microarray gene expression analysis was conducted with Affymetrix Mouse 430 2.0 GeneChips (Affymetrix Inc.).Following with gene knockdown, total RNA from spermatogonial stem cells was converted to cDNA. There are total 16 samples (Four groups and four samples per group) Negative control (N), Bcl6b Knockdown (B), Etv5 knockdown (E), and Pou3f1 knockdown (O), respectively.
Project description:Tumor progression is related to both genetic and epigenetic alterations. Until relatively recently, epigenetic changes were thought to target single genes only but we show that in Bladder tumours, epigenetic changes can affect whole chromosomal regions, resulting in the silencing of all the genes within those regions. This phenomenon is probably very general, and has been described in bladder, colon, breast and prostate cancers. In order to investigate epigenetic landscape and potential alterations in bladder, we established the chromatin profiling of RT112 cell line by ChIPseq for the following marks : H3K4me3, H3K9ac, H3K27me3, H3K9me3, H3K27ac, H3K4me1, and CTCF.
Project description:Tumor progression is related to both genetic and epigenetic alterations. Until relatively recently, epigenetic changes were thought to target single genes only but we show that in Bladder tumours, epigenetic changes can affect whole chromosomal regions, resulting in the silencing of all the genes within those regions. This phenomenon is probably very general, and has been described in bladder, colon, breast and prostate cancers. In order to investigate epigenetic landscape and potential alterations in bladder, we established the chromatin profiling of RT112 cell line by ChIPseq for the following marks : H3K4me3, H3K9ac, H3K27me3, H3K9me3, H3K27ac, H3K4me1, and CTCF.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare sh-NC with sh-Etv5 macrophage line Raw264.7 transcriptome profiling (RNA-seq).
Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro.
Project description:To elucidate the mechanism of ETV5-mediated pre-B cell survival signaling, we investigated the gene expression pattern in BCR-ABL1-transformed ETV5+/+ and ETV5-/- B cell precursors. BCR-ABL1 transformed B cell precursors of ETV5wild-type and ETV5 knockout mice were subjected to RNA isolation
Project description:The H3K27me3 ChIP-seq data for the human bladder transitional cell carcinoma cell line CL1207 were generated in order to detect regions of regional epigenetic silencing in this cell line and test the performance of several peak calling tools: CCAT (Xu et al., 2010) and HMCan (Ashoor et al., "HMCan – a tool to detect chromatin modifications in cancer samples using ChIP-seq data", submitted).
Project description:The H3K27me3 ChIP-seq data for the human bladder transitional cell carcinoma cell line CL1207 were generated in order to detect regions of regional epigenetic silencing in this cell line and test the performance of several peak calling tools: CCAT (Xu et al., 2010) and HMCan (Ashoor et al., "HMCan M-bM-^@M-^S a tool to detect chromatin modifications in cancer samples using ChIP-seq data", submitted). The human bladder cancer cell line CL1207 was derived from a muscle-invasive bladder cancer (De Boer et al., 1997). 5x105 cells were immunoprecipitated per ChIP assay with 4 M-NM-<g of rabbit polyclonal antibodies against trimethyl histone H3 lysine 27 (Upstate Biotechnology, Santa Cruz, CA) and DynabeadsM-BM-. Protein A (Invitrogen, Cergy Pontoise, France) in dilution buffer containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM TrisM-bM-^@M-^SHCl at pH 8.0, and protease inhibitors. Six ChIP assays in the same experimental conditions were necessary to perform one ChIP-Seq experiment, so the total of 3x106 cells for each of the duplicates.
Project description:In this research, Human OneArray Microarray analysis was performed to obtain broad spectrum information about the genes differentially expressed in human bladder cancer cell line RT112 and Gemcitabine Resistant Bladder Cancer cell line RT112-Gr.