Wilms Tumor-1 (WT-1) and its candidate target genes in gliomagenesis
ABSTRACT: WT-1 is a zinc-finger transcription factor with important roles during development. WT-1 is associated with the development of tumors of varied origins. Our lab has previously demonstrated aberrant expression of WT-1 in tumor cells. Necessity of WT-1 for tumor cell growth- in vitro & in vivo- was also shown. Overall design: Human glioma U251 cells were treated with Oligofectamine (CTRL), Oligofectamine + non-targeting siRNA control (SCR) or Oligofectamine+ si-WT1 siRNA (Si). Biological triplicates were run on microarrays.
INSTRUMENT(S): [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Project description:WT-1 is a zinc-finger transcription factor with important roles during development. WT-1 is associated with the development of tumors of varied origins. Our lab has previously demonstrated aberrant expression of WT-1 in tumor cells. Necessity of WT-1 for tumor cell growth- in vitro & in vivo- was also shown. Human glioma U251 cells were treated with Oligofectamine (CTRL), Oligofectamine + non-targeting siRNA control (SCR) or Oligofectamine+ si-WT1 siRNA (Si). Biological triplicates were run on microarrays.
Project description:BRD4 is a member of the bromodomain and extra-terminal (BET) family, which has become a promising drug target for numerous cancers. It has been reported that BRD4 is deregulated in gliomas; however, the precise molecular pathways regulated by BRD4 are still elusive. In this study, we knocked down BRD4 expression in glioma cell line U251 by lentivirus-mediated short hairpin RNA (shRNA) approach. BRD4 downregulation attenuated cell proliferation and promoted cell apoptosis. Genome-wide analysis of BRD4-regulated transcripts in U251 cells was performed using microarray to reveal the possible molecular mechanism Overall design: To knock down BRD4 expression in U251 cells, lentivirus that expressed shRNA against human BRD4 were transfected U251 cells. Scrambled shRNA (Scr-shRNA) that targeted a non-specific sequence was used as the normal control. The efficiency of BRD4 knockdown were demonstrated by real-time PCR and western blot assays. BRD4-shRNA- transfected and Scr-shRNA-transfected U251 cells were collected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Background: Wilms' tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia (AML). A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (KTS), is believed to change the DNA binding affinity of WT1. Altered balance between WT1 -KTS and WT1 +KTS expression associates with poor prognosis in AML. Methods: We characterized the DNA binding patterns of biotin-tagged WT1 -KTS and WT1 +KTS in K562 cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Results: We discovered that WT1 -KTS preferentially binds near transcription start sites (TSS) and in enhancers, whereas WT1 +KTS binds within gene bodies. Additionally, we observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif analysis showed enrichment of two TRANSFAC derived motif matrices within peaks for both isoforms, and enrichment of several previously published WT1 motifs which, however, differed between isoforms. Additional analyses showed that WT1 -KTS and WT1 +KTS target genes are transcribed to a higher extent than non-targets, and involved in cell proliferation, cell death, and development. Conclusions: Our results provide the first evidence that WT1 -KTS and WT1 +KTS bind principally different regions of the genome, yet share target genes. Our results indicate isoform-specific regulation of processes related to cell proliferation and differentiation, consistent with the involvement of WT1 in AML. Overall design: Streptavidin capture of biotinylated WT1 –KTS (two replicates), biotinylated WT1 +KTS (three replicates) and background K562-BirA cells (three replicates; background control). H3K4me3 immunoprecipitation in cells containing biotinylated WT1 –KTS (three replicates) and in cells containing biotinylated WT1 +KTS (two replicates).
Project description:Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1. Experiment Overall Design: Identification of WNT/Beta-Catenin or WT1 target genes. 39 individual samples.
Project description:The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that plays important roles during development of several organs including metanephric kidneys. A number of WT1 target genes have been identified, but the detailed mechanisms by which WT1 orchestrates renal development remain elusive. To identify WT1 target genes relevant to development, genome-wide expression profiling was performed using oligonucleotide microarrays representing 39,000 human transcripts Experiment Overall Design: Sample_source_name: UB27 cells, U2OS cells with Tetracycline-repressible WT1(-KTS) expression Experiment Overall Design: Sample_characteristics: U2OS cell line (osteosarcoma; female), Tetracycline-repressible WT1(-KTS) expression, time-course induction Experiment Overall Design: of WT1(-KTS) expression Experiment Overall Design: Sample_description: WT1(-KTS) expression was induced for 4, 8 or 12 Experiment Overall Design: hrs and the expression profile of each time-points was compared to the Experiment Overall Design: uninduced sample (0 hr).
Project description:Upstream regulator genes are central hub nodes in a network and their expression may response to upstream trigger factors of a disease and influence expression of hundreds of downstream genes, and further facilitate disease development. Therefore, the identification of upstream regulator genes are vital for understanding the pathophysiology of the disease and seek for potential therapeutic targets for the treatment of the disease. YAP1 was identified as a candidate upstream regulator for Alzheimer's disease (AD) in our study. To test whether expression alterations of YAP1 can lead to AD expression network disturbance and thus promoting AD progression, we knockdown and overexpressed YAP1 in U251 cell line with a stably expression of mutant APP (K670N/M671L) (U251-APP cells). RNA-seq (by IlluminaHiseq 4000) and following analyses were then performed to detect genes whose expression were influenced by YAP1 expression changes. The results indicated that expression alterations of YAP1 can significantly disturbe the whole AD expression network. Overall design: YAP1 inteference and overexpression were performed in U251 cell with a stably expression of mutant APP (K670N/M671L) (U251-APP cells) with three biological triplicates for each condition (siNC, si-YAP1, vector, and YAP1, one outlier for YAP1 overexpression was removed). Differential expression genes were obtained by comparing gene expression levels in YAP1 knockdown (si-YAP1) or overexpression (YAP1) cells with control cells (siNC or vector).
Project description:Analysis of gene expression levels in hematopoietic progenitor cells retrovirally transduced with full-length WT1(+/-) or a WT1-mutant lacking zinc-finger, WT1(delZ) cultured in vitro for 14 days. The hypothesis tested in this study was that the WT1-mutant confers an increased proliferation rate after 14 days in culture and an erythroid phenotype. Results provide information of upregulation of some genes associated with cellcycle progression, upregulated genes connected with erythropoiesis and downmodulation of some genes associated with myeloid differentiation. Total RNA obtained from CD34+ progenitor cells in suspension cultures for 14 days.
Project description:Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), is a rare autosomal recessive disorder characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor complex expressed on osteoclasts, dendritic cells, macrophages, monocytes, and microglia. At present, the precise molecular mechanisms underlying development of leukoencephalopathy and bone cysts in NHD remain largely unknown. We established THP-1 human monocyte clones that stably express small interfering RNA (siRNA) targeting DAP12 for serving as a cellular model of NHD. Genome-wide transcriptome analysis identified a set of 22 genes consistently downregulated in DAP12 knockdown cells. They constituted the molecular network closely related to the network defined by cell-to-cell signaling and interaction, hematological system development and function, and inflammatory response, where NF-kappaB acts as a central regulator. These results suggest that a molecular defect of DAP12 in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in NHD. The siRNA vector construct targeting the DAP12 sequence (SI) and the control vector construct targeting the scrambled sequence (SCR) were generated by using GeneClip U1 Hairpin cloning system (Promega). The vectors were transfected in THP-1 cells by using Lipofectamine LTX reagent. The stable cell lines were selected by incubating them for approximately two months in the feeding medium with inclusion of 200 microgram/ml Hygromycin B. Then, two SI clones named SI5 and SI17, in addition to two SCR clones named SCR1 and SCR4, were selected by limiting dilution of the cells in a manner of a single cell per well plated in a 96-well cell culture plate.
Project description:To elucidate the mechanism(s) underlying the synergistic interaction between ETV6 and NFKB1, we analyzed the genome-wide transcriptional consequences of single and double knock-downs of the two TFs in U251 cells. Overall design: The glioblastoma U251 cell line was transiently transfected with ETV6 and NFKB1 siRNA alone and then in combination, and were compared against control siRNA that doesn't target any known human transcript.