{"doi":"10.1101/2025.09.30.679661","title":"Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons","abstract":"Abstract 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 Abstract Introduction A 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) . Rationale Multiple 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 ","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":576564,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9452,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":554264,"name":"Kenneth Pham","orcid":"0000-0002-3352-9870","position":1,"is_corresponding":false},{"id":846399,"name":"Katelyn R. Titus","orcid":"0000-0002-8594-729X","position":2,"is_corresponding":false},{"id":1326683,"name":"Alexandria Nikish","orcid":"0000-0001-7320-4897","position":3,"is_corresponding":false},{"id":1369875,"name":"Constin Liu","orcid":null,"position":4,"is_corresponding":false},{"id":415777,"name":"Han-Seul Ryu","orcid":"0000-0001-9938-4902","position":5,"is_corresponding":false},{"id":1326684,"name":"Srikar S Muppidi","orcid":"0000-0003-1742-1584","position":6,"is_corresponding":false},{"id":1056362,"name":"Keerthivasan Raanin Chandradoss","orcid":"0000-0002-1990-0194","position":7,"is_corresponding":false},{"id":1460404,"name":"Peibo Xu","orcid":"0000-0001-7129-0445","position":8,"is_corresponding":false},{"id":1484982,"name":"R. Patel","orcid":"0009-0001-1873-7823","position":9,"is_corresponding":false},{"id":1056796,"name":"Ravi Boya","orcid":null,"position":10,"is_corresponding":false},{"id":401458,"name":"Jennifer E. Phillips‐Cremins","orcid":"0000-0002-4702-0450","position":11,"is_corresponding":false},{"id":1104292,"name":"Abraham J. Waldman","orcid":"0000-0002-4633-4415","position":0,"is_corresponding":true}],"reference_count":146,"raw_metadata":null,"created_at":"2026-07-19T02:57:56.636458Z","pmid":"41256638","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}