Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress
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ABSTRACT: Gene expression profile in CS1AN deficient and CSBwt restored cell lines after 24 hours of UV or alphe-amanitin treatment (only for restored). The comaprison of expression profile between 0 and 24 hours revealed continouse suppresion of transcription upon UV treatment in CS1AN cell line and alpha-amanitin treated CS1AN CSBwt restored cells.
Project description:Gene expression profile in CS1AN deficient and CSBwt restored cell lines after 24 hours of UV or alphe-amanitin treatment (only for restored). The comaprison of expression profile between 0 and 24 hours revealed continouse suppresion of transcription upon UV treatment in CS1AN cell line and alpha-amanitin treated CS1AN CSBwt restored cells. Gene expression profiles were obtained in duplicates for un-treated (assigned as 0h time point) and treated (assigned as 24h time point) cells.
Project description:Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.
Project description:Investigation of whole genome gene expression level changes in leaves of Evereste and MM106 genotypes 6 and 24 hours after infiltration (hpi) by Erwinia amylovora (Ea) versus mock (water + 0,01 % Silwet). This analysis include transcritome comaprison between the two apple genotypes just before inoculation (T0).
Project description:Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient's cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.
Project description:Cockayne syndrome B (CSB) protein is a member of the SWI/SNF family and has DNA-dependent ATPase and ATP-dependent chromatin remodeling activities. The CSB protein is missing or altered in CS-B cells. CS-B cells are hypersensitive to UV light and defective in transcription-coupled DNA repair (TCR). TCR efficiently removes a variety of lesions from the transcribed strand of active genes. It has been shown that lesions specifically in the transcribed strand of active genes trigger the induction of apoptosis following UV irradiation. Several DNA damage signaling cascades, including the ATR/Chk1, p38 kinase, p53, and jun N-terminal kinase pathways are activated following UV irradiation. However, the role of TCR in cellular global transcriptional responses to UV irradiation remains to be elucidated. Using oligonucleotide microarray technology, we analyzed the time course of responses of CS-B cells (CS-B) and CS-B cells complemented with wild-type CSB cDNA (CS-B wt). Experiment Overall Design: In order to investigate the global transcriptional responses to UV damage in TCR-proficient or TCR-deficient cells, CS-B wt and CS-B cells were irradiated with 10 J/m2 of UV light and incubated for 2 or 12 hours. All experiments were performed in triplicate.
Project description:The transcriptional response to 10 J/m2 of UV-light (254 nm) was assessed with HGU95AV2 Affymetrix probe arrays at 6, 12, 18 and 24 hours after exposure. Mock-treated samples were assessed at 6 and 24 hours. Keywords: time-course
Project description:The transcriptional response to 50 J/m2 of UV-light (254 nm) was assessed with HGU95AV2 Affymetrix probe arrays at 6, 12, 18 and 24 hours after exposure. Mock-treated samples were assessed at 6 and 24 hours. Keywords: time-course
Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours
Project description:Cockayne syndrome is an inherited premature aging syndrome associated with developmental and neurological disorders. Mutations in the genomic locus encoding CSB are associated with 80% Cockayne syndrome cases. CSB is invovled in relieving UV-induced and oxidative stree. To gain more insights into the fucntion of CSB under these stress, we use ChIP-seq to determine the genomic localization of CSB 1 hour after UV irradiation and menadione treatment.
Project description:Cockayne syndrome is an inherited premature aging syndrome associated with developmental and neurological disorders. Mutations in the genomic locus encoding CSB are associated with 80% Cockayne syndrome cases. CSB is invovled in relieving UV-induced and oxidative stree. To gain more insights into the fucntion of CSB under these stress, we use ChIP-seq to determine the genomic localization of CSB 1 hour after UV irradiation and menadione treatment. Genomic localization of CSB and remodeling deficient CSBâN1