Pre-occupied ATF4 relies on CEBPγ co-binding for robust transcription activation during integrated stress response [HeLa_CCCP_CUT&Tag]

Upon encountering stress, cells need to rapidly and extensively remodel their transcriptome to restore homeostasis and ensure survival. However, the molecular mechanisms underlying stress-induced transcriptional remodeling are not well understood. In this study, we employed multi-omics approaches to comprehensively profile the changes in transcriptome, epigenetic landscapes, and 3D genome organization during the integrated stress response (ISR), a conserved cellular pathway activated by diverse stress stimuli. Our findings reveal that widespread transcriptional changes occur 4-6 hours after the induction of ISR, coinciding with the genome-wide binding of key transcriptional effector ATF4. Notably, while ATF4 binds to most of its target genes only upon ISR activation, it also occupies the promoters of hundreds of genes under non-stress conditions, priming them for enhanced transcriptional activation in response to stress signals. We further demonstrate that ATF4-mediated gene activation does not rely on traditional transcriptional regulatory mechanisms, such as increased H3K27 acetylation, chromatin accessibility, or remodeling of enhancer-promoter loops. Instead, our data suggest that the nuclear localization of ATF4 induces a pronounced genomic redistribution of transcriptional factor CEBP-γ, from non-ATF4 sites to a subset of ATF4-bound regions, where it collaborates with ATF4 to elicit transcriptional activation. The combined occupancy of ATF4 and CEBP-γ ultimately dictates the transcriptional outcomes during ISR. Interestingly, the preferred binding sites of CEBP-γ in response to stress are not determined by the strength of the CEBP-γ or ATF4 motifs, but rather by the higher-order chromatin architecture surrounding the binding sites. These findings provide novel insights into the molecular mechanisms that govern the rapid and efficient transcriptional remodeling during ISR, with potential implications for other stress responses.