Temporal Coordination of the Transcription Factor Response to H2O2stress [scATAC-seq and scRNA-seq]

The p53 and FOXO transcription factors (TFs) share many similarities despite their distinct evolutionary origins. Both TFs are activated by a variety of cellular stresses and upregulate genes in similar pathways including cell-cycle arrest and apoptosis. Oxidative stress from excess H2O2activates both FOXO1 and p53, yet whether they are activated at the same time is unclear. Here we found that cells respond to high H2O2levels in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 levels remain low. In the second phase FOXO1 exits the nucleus and p53 levels rise. We found that other oxidative stress induced TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN) but not both following H2O2stress. The two TF phases result in large differences in gene expression patterns. Finally, we provide evidence that 2-Cys peroxiredoxins control the timing of the TF phases in response to H2O2. MCF7 cells were plated (50,000 cells/well) on plastic 6 well plates and allowed to attach for 48 hours. Cells were then treated with PBS control, 50 µM H2O2, and 75 µM H2O2 for 5 hours. Nuclei were then isolated using the Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression protocol (CG000365). All further steps with the kit were performed by the University of Arizona Genetics Core. Sequencing was carried out using Novogene. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (Product code: 1000285) from 10X Genomics was used for transposition, GEM generation and Barcoding, ATAC Library Construction, cDNA Amplification, Gene expression and library construction (CG000338). Please note that processed data files were generated from all 16 files (linked to each sample records) together using 10X genomics cellranger -arc program