Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons

Persistent molecular correlates of long-term memory storage remain an open question. Here, we stimulate and re-stimulate human neurons and use multi-modal single-nucleus technologies to query DNA methylation, higher-order chromatin folding, and gene expression. We find enduring traces of activity-gained and activity-lost chromatin loops. Genes anchoring persistent activity-gained loops exhibit activity-upregulated expression, whereas persistent activity-lost loops anchor activity-downregulated genes that remain repressed five days post-stimulation. CTCF-bound looped enhancers and promoters are refractory to activity-dynamic DNA methylation. Looped enhancers bound by CTCF can exhibit memory of activity-induced histone modifications and persistent expression of activity-upregulated genes. Upon second stimulation, activity-upregulated genes are robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. Activity-independent gene expression can be downregulated when unlooped but protected from homeostatic downscaling when anchored in persistent loops. Our data reveal long-term genome folding persistence linked to plasticity of activity-dependent gene expression during recall in human neurons. Structured AbstractO_ST_ABSIntroductionC_ST_ABSA long-standing question in neuroscience is how memories of previous experiences are stored in the mammalian brain to facilitate recall over the lifetime of an individual. Classic models of memory posit that both synapse-specific events and cell-wide transcriptional programs are required for encoding, consolidation, and long-term storage of memory (1, 2). Learning involves synapse strengthening, and synapse weakening has been linked to memory loss (3, 4). Nascent transcription and protein synthesis also occur in response to neural activation in vitro and in vivo (5-7). A leading hypothesis, the synaptic tagging and capture model, asserts that specific synapses are biochemically marked during encoding and newly made cell-wide RNA/proteins act specifically on tagged synapses to maintain long-term memory (8). Multiple historic studies have pursued the identification of proteins linked to potentiated synapses (9-13), but few explore the possibility of persistent, activity-dependent patterns of DNA, chromatin, higher-order chromatin folding, or RNA in human models of long-term memory. Over the last decade, the molecular technique of Chromatin-Conformation-Capture has been employed to discover that the mammalian genome folds into thousands loops of (5, 14-28). Loops bring distal non-coding cis regulatory elements into contact with their target genes to influence gene expression (29). They form via the processive motion of the cohesin ring along chromatin until it stalls at the architectural protein CTCF, thus extruding out the intervening DNA (22, 30-32). Loops can markedly reconfigure during lineage commitment in development and in response to genetic perturbations, and their formation is critical for spatiotemporal regulation of expression (22, 30, 33-41). RationaleMultiple recent works suggest that loops connecting activity-dependent enhancers to distal genes and can be induced during neural stimulation in vitro and behavior paradigms in vivo (5, 27, 42, 43). Genetic elimination of the architectural proteins CTCF and cohesin prior to learning substantially impairs memory encoding across multiple behavior tasks in vivo (42, 44, 45). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo and activity-dependent gene expression during neural stimulation in vitro (27). Fear conditioning recruits epigenetically plastic neurons to form the memory engram (46) and induces chromatin accessibility changes at enhancers that might persist at least five days after fear conditioning (43). Together, these data provide the rationale for our hypothesis that key structural features of higher-order chromatin folding could be persistent long after the exposure and removal of a pharmacological, environmental, or behavioral stimulus that causes neurons to fire action potentials. ResultsHere, we apply cutting-edge single-nucleus multi-modal technologies toward the goal of ascertaining the extent to which there are enduring traces of higher-order chromatin folding, DNA methylation, histone modifications at activity-dependent enhancers, and/or mRNA levels in single human neurons upon exposure to a repetitive stimulation and re-stimulation paradigm. We find that pharmacological stimulation of human induced pluripotent stem cell (iPSC)-derived neurons induces chromatin loop plasticity, including persistent activity-gained (PG) and persistent activity-lost (PL) loops that are dependent on CREB phosphorylation. We also query activity-dependent patterns of DNA methylation genome-wide and demonstrate that the majority of looped enhancers and promoters are negligibly differentially methylated in human neurons in response to pharmacological stimulation. CTCF binding to looped enhancers and promoters further protects such regulatory elements from activity-stimulated changes in DNA methylation. What are the potential functional effects of persistent chromatin loops? We find that activity-lost persistent loops anchor activity-downregulated genes that remain repressed five days post-stimulation. By contrast, activity-gained persistent loops are enriched for activity-induced gene expression at 2-5 hours post-stimulation, but mRNA levels resume baseline levels by five days after removal of the stimulus. A subset of promoter-enhancer loops bound by CTCF can exhibit enduring traces of the histone modification H3K27ac. Promoters persistently looped to CTCF-bound persistent activity-induced enhancers can exhibit memory of activity-upregulated gene expression for at least five days after stimulation. Finally, we re-stimulated our human neurons five days after the original stimulation event and assessed gene expression with single-nucleus RNA-seq. Upon second stimulation, we unexpectedly find that activity-upregulated genes can be robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. We also unexpectedly find that activity-independent, invariant gene expression can be downregulated at second stimulation when unlooped but protected from homeostatic downscaling when anchored in persistent loops. ConclusionTaken together, our data reveal enduring traces of higher-order chromatin loops in human neurons after activity-simulation and their link to gene expression plasticity and habituation during re-stimulation and re-stimulation of human neurons. Our work sheds light on the role for chromatin and gene expression in an important unsolved paradox in neuroscience: How is information encoded in neural circuits on the timescale of years despite the rapid turnover of synaptic proteins/RNAs in hours to weeks? Persistent chromatin loops shape plasticity in cell-wide RNA levels, thus laying the foundation for future studies linking genes identified here to their subcellular localization and possible functional role at the synapse.