Project description:Oxygen is essential for cell growth, however in tumours quick growth can cause hypoxic conditions. To determine the changes in the chromatin landscape in response to hypoxia in breast cancer we have performed ATAC-seq dugin normoxia and hypoxia.
Project description:Purpose: Study hypoxia induced changes in genome-wide H3K27me3 occupancy Methods: Using the MCF7 breast epithelial adenocarcinoma cell line as a model, we studied epigenomic reprogramming as a function of fluctuating oxygen tension. To this end, we combined chromatin-immunoprecipitation and deep-sequencing analysis to identify H3K27me3-marks in MCF7 cells subjected to changes in oxygenation (i.e. acute hypoxia, chronic hypoxia). Results: H3K27me3-marks showed a rapid global increase at specific sites throughout the genome under hypoxia, both genic and inter-genic. Conclusions: Our data show that oxygen availability dynamically regulates the epigenetic state of the genome.
Project description:Purpose: Study hypoxia induced changes in genome-wide H3K27me3 occupancy Methods: Using the MCF7 breast epithelial adenocarcinoma cell line as a model, we studied epigenomic reprogramming as a function of fluctuating oxygen tension. To this end, we combined chromatin-immunoprecipitation and deep-sequencing analysis to identify H3K27me3-marks in MCF7 cells subjected to changes in oxygenation (i.e. acute hypoxia, chronic hypoxia). Results: H3K27me3-marks showed a rapid global increase at specific sites throughout the genome under hypoxia, both genic and inter-genic. Conclusions: Our data show that oxygen availability dynamically regulates the epigenetic state of the genome. Genome-wide H3K27me3-mark profiles were generated by combining ChIP analysis with deep sequencing using Illumina GAIIx.
Project description:Purpose: Study hypoxia and reoxygenation induced changes in genome-wide gene expression Methods: Using the MCF7 breast epithelial adenocarcinoma cell line as a model, we studied epigenomic reprogramming as a function of fluctuating oxygen tension. To this end, we performed a transcriptomics analysis in MCF7 cells subjected to changes in oxygenation (i.e. acute hypoxia, chronic hypoxia, reoxygenation). Results: Global downregulation upon hypoxia; partial restore on reoxygenation. Conclusions: Our data show that oxygen availability dynamically regulates gene transcription.
Project description:Purpose: Study hypoxia and reoxygenation induced changes in genome-wide H3K4me3 and H3K27me3 occupancy Methods: Using the MCF7 breast epithelial adenocarcinoma cell line as a model, we studied epigenomic reprogramming as a function of fluctuating oxygen tension. To this end, we combined chromatin-immunoprecipitation and deep-sequencing analysis to identify H3K4me3-marks and H3K27me3-marks in MCF7 cells subjected to changes in oxygenation (i.e. acute hypoxia, chronic hypoxia, reoxygenation). Results: H3K4me3 and H3K27me3-marks showed a rapid global increase at specific sites throughout the genome under hypoxia, both genic and inter-genic, that was partly restored upon reoxygenation. Conclusions: Our data show that oxygen availability dynamically regulates the epigenetic state of the genome.
Project description:We report the generation of CRISPR/Cas9-engineered MCF7 human breast cancer cells that lack functional HIF1α. We show that the HIF1α-dependent transcriptional response to hypoxia is severely impaired in HIF1α-mutant MCF7 cells.
Project description:Background: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins. Methods: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays. Results: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production Conclusion: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs. MCF7 cells were treated with three different conditions. Treatment-1: MCF7 cells were exposed to hypoxia (0.1% O2) for 48 h and harvested for RNA extraction (n=3). Treatment-2: MCF7 cells were exposed to normoxia for 48 h and harvested for RNA extraction (n=3). Treatment-3: Dicer inhibition in MCF7 cells by transient transfection of siRNAs targeting Dicer. Cells were transfected with 20 nM siRNA duplexes (Shanghai GenePharma Co., Ltd, China), using Lipofectamine 2000 reagent (Invitrogen) following the manufacturerM-bM-^@M-^Ys protocol. A second transfection was carried out after 24 h following the same protocol. Cells were harvested 24 h after the second transfection and used for RNA extraction (n=3). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Affymetrix miRNA 3.1 Array Strip was used for RNA analysis. This array consisted probe sets unique to human mature and pre-miRNA hairpins. A detailed protocol can be found in the miRNA 3.1 Array Strips technical manual (Affymetrix). In summary, 100-300 ng of total RNA was used to synthesise double stranded cDNA using random hexamers. The cDNA was then amplified to produce antisense cRNA, which was then reverse transcribed in a second cycle of cDNA synthesis. The second cycle incorporates dUTP into the cDNA sequence, which allows it to be fragmented using uracil DNA glycosylase and apurinic/apyrimidic endonuclease I. Following biotinylation, these fragments were hybridised overnight to a Affymetrix miRNA 3.1 array. The arrays were then washed, stained using a fluorescently-labelled antibody, and scanned using a high-resolution scanner. Intensity data were analysed using PartekM-BM-. software (Partek Inc.). Data were normalised by quantile normalisation and log 2 transformed. Differential expression was determined by ANOVA and corrected for false discovery.
Project description:Regulation of gene expression by the CtBP family of NADH-sensitive transcriptional regulators, in MCF7 cells under normoxia and hypoxia. To determine the effect of CtBP knockdown on gene expression in MCF7 we transfected cells with an siRNA (5′-GGGAGGACCUGGAGAAGUUdTdT-3′/3′-dTdTCCCUCCUGGACCUCUUCAA-5′, obtained from Ambion) targetting both CtBP1 and CtBP2 (versus control siRNA). After 48 hours cells were either transferred to a hypoxic chamber (1% oxygen), or maintained in normoxia, for 18 hours.