Induction of hysteretic chromatin states by CRISPRa reveals locus-specific encoding of transcriptional memory

Transcriptional memory is a phenomenon that allows cells to 'remember' prior stimulatory history, which can alter subsequent gene expression patterns and shape development, immunity, and disease. Although various epigenetic adaptations have been implicated, it remains unclear whether the mechanics of transcription alone are sufficient to install durable memory independently of complex activation and global phenotypic changes induced by inflammatory and environmental stimuli. Here, we employ CRISPR activation (CRISPRa) to uncouple gene activation from global signaling and test whether memory can be established selectively by a local transcriptional pulse in primary mesenchymal stromal cells (MSCs). Key stimulus-responsive genes (IDO1, CXCL11) remain sensitized for weeks after either IFN{gamma} exposure or locus-specific CRISPRa perturbations. Histone PTM profiling links this memory to epigenetic hysteresis involving persistent loss of promoter H3K27me3. Single-molecule chromatin mapping by DAF-seq showed that this adaptation does not coincide with gains in promoter accessibility, but suggests it supports increased transcription factor reacquisition in rechallenged cells. Finally, while H3K27me3 loss appears to be a key hallmark of strongly induced memory genes, multi-omic analyses showed that persistent H3K27me3 hysteresis also occurs at a small subset of non-memory genes. Coordination of H3K27me3 hysteresis with additional cis factors therefore likely shapes transcriptional memory in a locus-specific fashion, underscoring the utility of targeted CRISPRa perturbations for dissecting these responses.