Topoisomerase I cleavage complexes preferentially down-regulate ubiquitin- and RNA degradation-related transcripts in relationship to gene length, exon-intron junctions and p53-dependent up-regulation of miR-142-3p
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Michael Ryan
PROVIDER: E-GEOD-37358 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:DNA topoisomerase I (Top1) is required for transcription as it relaxes positive and negative supercoils by forming transient Top1 cleavage complexes (Top1cc) up- and down-stream of transcription complexes. However, Top1cc can also be trapped by endogenous DNA lesions and by camptothecin (CPT) and its anticancer derivatives, which results in transcription blocks. Here, we undertook a genome-wide analysis of the effects of CPT on gene expression at exon resolution. We tested the impact of Top1 inhibition on gene expression at the genome-wide level in human colon carcinoma HCT116 and human breast carcinoma MCF7 cells. The RNA of cells treated with camptothecin (CPT) for various times was analyzed with Affy Exon array (GeneChip Human Exon 1.0 ST array). Moreover, we tested the impact of Top1 down-regulation on gene expression at the genome-wide level in human colon carcinoma HCT116 cells. The RNA of cells treated transfected with a Top1 siRNA was analyzed with Affy Exon array (GeneChip Human Exon 1.0 ST array).
2013-04-17 | E-GEOD-37352 | biostudies-arrayexpress
Project description:DNA topoisomerase I (Top1) is required for transcription as it relaxes positive and negative supercoils by forming transient Top1 cleavage complexes (Top1cc) up- and down-stream of transcription complexes. However, Top1cc can also be trapped by endogenous DNA lesions and by camptothecin (CPT) and its anticancer derivatives, which results in transcription blocks. Here, we undertook a genome-wide analysis of the effects of CPT on gene expression at exon resolution. We tested the impact of Top1 inhibition on miRNA expression at the genome-wide level in human colon carcinoma HCT116. The miRNA of cells treated with camptothecin (CPT) for were measured at several timepoints with Agilent Human miRNA Microarray V3 (8x15K).
2013-04-17 | E-GEOD-37357 | biostudies-arrayexpress
Project description:The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1siRNA cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those cells, Top2 compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-H2AX foci associated with replication. siTop1 cells also show rDNA and nucleolar alterations, and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) was reduced in siTop1 cells, as it also was in cells with transient Top1 downregulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacological profiling showed l-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, aphidicolin, hydroxyurea and staurosporine, and hypersensitivity to etoposide and actinomycin D demonstrated that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability and apoptosis. Overall, our studies demonstrate the pleiotropic nature of human Top1 activities. In addition to its classical DNA nicking-closing functions, Top1 plays critical non-classical roles in genomic stability, gene-specific transcription, and response to various anticancer agents. Experiment Overall Design: We are using Affymetrix whole genome array U133 Plus 2, which is a single color microarray platform. For colon experiments, we analyzed 2 different samples as 4 replicates. For breast experiments, we analyzed 2 samples as replicates.
2008-06-15 | E-GEOD-7161 | biostudies-arrayexpress
Project description:Topoisomerase I (Top1) relaxes both positive and negative supercoilings by producing transient Top1 cleavage complexes (Top1cc). Several studies have suggested the implication of Top1 in splicing. Here, we tested the implication of Top1cc in splicing at the global genome level in human carcinoma cells to determine whether Top1 inhibition selectively affects particular families of genes. We tested the impact of Top1 inhibition on splicing at the genome-wide level in human colon carcinoma HCT116 cells. The RNA of HCT116 cells treated with camptothecin (CPT) for various times was analyzed with ExonHit Human Splice Array.
2011-07-21 | E-GEOD-23677 | biostudies-arrayexpress
Project description:Within the overall project, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (pro)renin receptor ((P)RR), stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. In this dataset, we include the expression data obtained from siRNA against (P)RR in KELLY cells.Three intervention samples and three control samples were analyzed. We generated the following pairwise comparisons using Chipinspector (Genomatix Software GmbH) and a FDR of 2%. ChipInspector carries out significance analysis on the single probe level. Normalized probe set level data not provided for individual Sample records. Processed data is available on Series record.
2015-08-07 | E-GEOD-39963 | biostudies-arrayexpress
Project description:Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. siRNA knockdown of RBM38 was perfomed in human MCF-7 breast cancer cells. The efficiency of RBM38 knockdown was monitored by western blot using an RBM38 antibody (Santa Cruz Biotechnology, SC-85873). We conducted HJAY exon and exon junction array profiling on RNAs from four siRBM38 treated MCF-7 samples vs. four sicontrol treated MCF-7 samples Control / knockdown comparison.
2013-07-29 | E-GEOD-49293 | biostudies-arrayexpress
Project description:The aim of this experiments was to characterise the transcriptomic changes occurring in cancer cells, in which ZNF84 gene is silenced, in control conditions and when cells undergo stress by being treated with cytostatic doses of doxorubicin. We anticipated that ZNF84 regulates the level of p21 cell cycle inhibitor and therefore contributes to senescence induction in response to stress. We wanted to know whether attenuation of senescence in cells with downregulated ZNF84 is accompanied by alterations in other biological processes and signalling pathways. Cells were transfected with 30 nM siRNA (against ZNF84 or negative control siRNA) and then either a) doxorubicin was added (48 hours after transfection and cells were cultured in the presence of the drug for another 48 hours) b) no drug was added (cells were cultured in drug-free medium for 72 hours post- transfection) Variants a) and b) were separate RNA sequencing experiments.
2021-05-05 | E-MTAB-9848 | biostudies-arrayexpress
Project description:The cancer target enyzme topoisomerase 1 transiently cleaves one strand of chromosomal DNA to relax accumulated strain that can prevent transcription, replication or chromatin assembly. The topoisomerase 1 poison camptothecin and its synthetic analogs are widely used chemotherapeutics that act by trapping the enzyme on DNA in a ‘covalent complex’, resulting in persistent DNA damage and cell death. The prevailing model, interfacial inhibition, contends that the covalent complex is trapped by drug binding alone. In contrast, here we show that camptothecins produce extensive oxidative stress and that topoisomerase 1 is reactive with electrophilic second messengers of oxidative damage. We show that blocking oxidative stress in cells inhibits covalent complex formation by camptothecin, while electrophiles such as 4-hydroxynonenal are able to induce covalent complex formation in cells on their own. Towards mechanism of action, we show that 4-hydroxynonenal electrophilically modifies a cysteine within the DNA-binding active site of human topoisomerase 1. Taken together, our results suggest a mechanism in which oxidative stress mediates the effects of many topoisomerase 1 poisons, and suggest that this critical DNA-regulating enzyme may have a dual role as a sensor of cellular redox stress, linking oxidative stress to DNA damage response in cancer cells.
2021-03-24 | PXD018549 | Pride
Project description:HCT116 cells were transfected with two different siRNA's targeting either DDX5, an siRNA targeting EBNA1, or no siRNA (mock). The siRNA targeting EBNA1 is used as a negative control since HCT116 cells do not have the EBNA1 gene. RNA was obtained from cultures at 24hrs post-siRNA transfection using the Qiagen RNeasy Minikit (cat. # 74104) with on-column DNase digestion performed as per the manufacturer's protocol. The RNA samples were isolated at 24hrs post-siRNA transfection since this timepoint precedes an impaired G1-to-S phase cell cycle progression phenotype that is evident at 48hrs post-siRNA transfection and so may reveal gene expression changes occuring before this effect on cell cycle. RNA samples were submitted to the Cold Spring Harbor Laboratory Microarray Faciity where cDNA was prepared, labeled, and hybridized to Affymetrix GeneChip Human Gene 1.0 ST microarrays. Data from the arrays were processed using the RMA method with an up-to-data probe set definition (Biostatistics 4:249-264 and Nucleic Acids Research 33(20):e175. Gene set analysis was performed using generally applicable gene set enrichment (BMC Bioinformatics 10:161). The most differentially regulated gene ontology groups were selected with FDR q-value < 0.1. Expression data from 3 independent HCT116 cutures transfected with either DDX5si2008, DDX5si2053, or EBNA1si1666 and 2 independent cultures transfected with mock conditions (no siRNA) were obtained from RNA samples prepared 24hrs post-siRNA transfection. These cultures were seeded with 200,000 cells per well 24hrs prior to siRNA transfection and were grown in DMEM media supplemented with 10% fetal bovone serum. Transfections were performed using lipofectamine RNAiMAX (Invitrogen 13778-075) and the manufacturer's protocol.
2012-06-01 | E-GEOD-36141 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE34905: Differential gene expression caused by Dicer knockout in small intestine GSE34909: Differential miRNA expression caused by Dicer knockout in small intestine Refer to individual Series
2012-01-07 | E-GEOD-34910 | biostudies-arrayexpress