Project description:Lactate enable to cause a novel post-translational modification, lactylation of proteins. We are interested in exploring whether lactate modulates DNA-damaging agents resistance in the form of lactylation. To gain a global view of DNA-damaging agents resistance-related lactylation, especially Kla of nonhistone substrates, we used 4D-Label free high-resolution LC-MS/MS, quantitative lysine lactylation analysis to investigate Kla substrates in cisplatin-resistant AGS cells.
Project description:Lactate enable to cause a novel post-translational modification, lactylation of proteins. We are interested in exploring whether lactate modulates DNA-damaging agents resistance in the form of lactylation. To gain a global view of DNA-damaging agents resistance-related lactylation, especially Kla of nonhistone substrates, we used 4D-Label free high-resolution LC-MS/MS, quantitative lysine lactylation analysis to investigate Kla substrates in cisplatin-resistant AGS cells.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. We identified that depletion of KG sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. KG is required for the activity of KG-dependent dioxygenases (KGDDs), and prior work has shown that changes in KGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 KGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, KG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation. The increase in histone acetylation via the KG-carnitine axis promoted HR-mediated DNA repair through site-specific histone acetylation. Finally, we observed a positive correlation between TMLHE and histone acetylation in patient samples and found that high TMLHE or acetylcarnitine correlates with worse progression free survival in patients treated with DNA damaging agents. This study demonstrates for the first time KG affects histone acetylation and provides a mechanism of HR-proficiency via carnitine synthesis. Moreover, these data provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.
Project description:Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.
Project description:Overexpression of the transcription factor RHOXF2 in the HEK293_M2 cells confers resistance to several DNA damaging agents. To understand that resistance mechanism, we cultured HEK293_M2 with or without overexpression of RHOXF2 in the presence of 40nM of mitomycin C or DMSO (control) and profiled their transcriptional response by RNA-seq.
