Project description:Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to cell cycle arrest and apoptosis. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Characterizing genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT. O6-MeG signatures were highly similar to mutational signatures from patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes in TMZ-exposed cells and naked DNA proposing an indirect influence of transcription to O6-MeG formation. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and nucleotide sequence. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.
Project description:Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to apoptosis due to mismatch-repair overload. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair mechanisms, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT transfection. O6-MeG signatures were highly similar to mutational signatures of patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes, suggesting an additional transcription-associated contribution to its repair. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and regulation. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.
Project description:Whole genome gene expression analysis was examined with Ralstonia eutropha strain H16 cultures grown in PHB production medium (recipe per Peoples and Sinskey, 1989) containing fructose or trioleate as the main carbon source. The goal of this analysis was to determine the identity of the triacylglycerol and fatty acid breakdown genes in R. eutropha strain H16.
Project description:Whole genome gene expression analysis was examined with Ralstonia eutropha strain H16 cultures grown in PHB production medium (recipe per Peoples and Sinskey, 1989) containing fructose or trioleate as the main carbon source. The goal of this analysis was to determine the identity of the triacylglycerol and fatty acid breakdown genes in R. eutropha strain H16. In the study presented here, triplicates of R. eutropha strain H16 were examined for changes in expression of 6702 genes during growth and PHB production on each carbon source.
Project description:Ralstonia eutropha H16 is well-known to produce poly(3-hydroxybutyrate) [P(3HB)], a kind of polyhydroxyalkanoates attracted as bio-based biodegradable plastics, efficiently as an energy storage material under unbalanced growth conditions. To obtain further extended knowledge of PHA biosynthesis, this study employed quantitative transcriptome analysis based on deep sequencing of complementary DNA generated from RNA (RNA-seq) for R. eutropha H16. Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined by using Illumina high-throughput sequencer. The RNA-seq results supported induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in growth phase, and repression trends for genes involved in central metabolisms in PHA production phase. Interestingly, transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several selected genes for ?-oxidation were significantly induced in PHA production phase even when the cells were grown on fructose. mRNA profiles of R. eutropha H16 grown on fructose at different phases were generated by deep sequencing, in duplicate, using Illumina GAIIx.