{"ID": "PMID:39626660", "aka": "https://www.ncbi.nlm.nih.gov/pubmed/39071420", "lab": {"uuid": "312cb909-76a6-405d-a96c-c3292abf08a1", "@id": "/labs/alistair-boettiger-lab/", "status": "current", "@type": ["Lab", "Item"], "display_title": "Alistair Boettiger, STANFORD", "title": "Alistair Boettiger, STANFORD", "correspondence": [{"contact_email": "YWJvZXR0aWdAc3RhbmZvcmQuZWR1", "@id": "/users/b8835c78-05e3-4173-a6c5-1ab93b4d12cc/", "display_title": "Alistair Boettiger"}], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.312cb909-76a6-405d-a96c-c3292abf08a1"]}}, "url": "https://www.ncbi.nlm.nih.gov/pubmed/39626660", "award": {"project": "4DN", "@id": "/awards/1U01DK127419-01/", "description": "RT-CDF: Chromatin structure and transcription regulation are essential for cellular function, and their dynamics are highly correlated both in development and in disease. However, despite decades of amazing work identifying the molecular players involved in these processes, and mapping their interactions genome-wide, we are currently unable to describe the function connecting 3D chromatin structure and transcription dynamics. This limitation stems from the fact that chromatin structure and gene expression emerge from intrinsically stochastic transitions at the single-cell level, and we are missing the critical temporal parameters associated with these transitions. Therefore, new tools to measure both chromatin structure and transcription over time in single cells are critical for understanding how the human genome is read and for predictively controlling the epigenome.  Here, we propose to develop a new set of live single-cell imaging technologies to simultaneously measure changes in 3D chromatin structures and their associated dynamics of gene expression across a large range of timescales: from dynamics of individual topologically associated domains and enhancer-promoter interactions, to changes associated with stable epigenetic memory across cell cycles. For the shorter timescales (under a cell cycle), our new imaging approach combines live super-resolution microscopy of fluorescently labeled loci with end-point demultiplexing of loci identity using Optical Reconstruction of Chromatin Architecture (ORCA), in order to track and trace 3-12 points within a functional chromatin unit. This new technique, which we call live-ORCA, will allow us to measure for the first time the temporal dynamics of an entire topologically associated domain in single cells. We will use live-ORCA in conjunction with time-lapse imaging of transcriptional bursting to study the dynamics of promoter-enhancer activity throughout cell differentiation and under perturbations of the chromatin network. For the longer timescale (across multiple cell cycles), our approach will combine time-lapse microscopy of gene expression, monitoring the distance between two tagged genomic loci as a live reporter of chromatin structure, and end-point chromatin tracing of the entire gene neighborhood using ORCA. We will perform these measurements in two systems: at a highly controlled synthetic reporter where we can induce either short-term silencing or long-term epigenetic memory, and at time points in differentiation when genes commit epigenetically to a new transcriptional state. Moreover, in order to further investigate the mechanism of epigenetic inheritance, we will develop a novel microfluidic device that allows us to track changes in chromatin 3D structures across individual cell lineages. Finally, to test our quantitative understanding, we will go back and forth between these single-cell data and theoretical modelling of chromatin dynamics. This research plan will greatly advance our understanding of chromatin dynamics and its functional role in transcription regulation, while at the same time contributing a whole new set of novel imaging technologies and engineered cell lines that will serve as a jumping board for the 4D Nucleome and broader scientific community.", "status": "current", "@type": ["Award", "Item"], "center_title": "Bintu", "display_title": "LIVE-CELL MULTIPLEX SUPER-RESOLUTION IMAGING OF CHROMATIN STATE TRANSITIONS", "uuid": "b7c5d0c8-053e-4da3-b446-b68787a5a738", "name": "1U01DK127419-01", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "title": "Long-range regulation of transcription scales with genomic distance in a  gene-specific manner.", "status": "current", "authors": ["Jensen CL", "Chen LF", "Swigut T", "Crocker OJ", "Yao D", "Bassik MC", "Ferrell JE Jr", "Boettiger AN", "Wysocka J"], "journal": "Molecular cell", "abstract": "Although critical for tuning the timing and level of transcription, enhancer  communication with distal promoters is not well understood. Here, we bypass the  need for sequence-specific transcription factors (TFs) and recruit activators  directly using a chimeric array of gRNA oligos to target dCas9 fused to the  activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective  activator recruitment to arbitrary genomic sites, even those inaccessible when  targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in  mouse embryonic stem cells (mESCs), where neither gene is expressed. Although  activator recruitment to any tested region results in the transcriptional  induction of at least one gene, the expression level strongly depends on the  genomic distance between the promoter and activator recruitment site. However,  the expression-distance relationship for each gene scales distinctly in a manner  not attributable to differences in 3D contact frequency, promoter DNA sequence,  or the presence of repressive chromatin marks at the locus.", "date_created": "2025-08-12T17:05:24.683021+00:00", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2025-08-12T17:05:24.870191+00:00"}, "date_published": "2025-01-16", "public_release": "2025-08-12", "schema_version": "2", "project_release": "2025-08-12", "exp_sets_prod_in_pub": [{"status": "released", "experimentset_type": "replicate", "accession": "4DNESPB3DGGV", "@type": ["ExperimentSetReplicate", "ExperimentSet", "Item"], "display_title": "4DNESPB3DGGV", "@id": "/experiment-set-replicates/4DNESPB3DGGV/", "uuid": "8c75374e-c575-41c0-9a1a-3b090a2a79c8", "experiments_in_set": [{"uuid": "254f0951-b89e-4337-8ce0-0e2325ee2486", "@type": ["ExperimentMic", "Experiment", "Item"], "display_title": "multiplexed FISH on R1 mESC line - 4DNEXEZBQPKD", "status": "released", "@id": "/experiments-mic/4DNEXEZBQPKD/", "experiment_type": {"@type": ["ExperimentType", "Item"], "status": "released", "@id": "/experiment-types/multiplexed-fish/", "display_title": "multiplexed FISH", "title": "multiplexed FISH", "uuid": "bcdda46a-489d-4d22-be80-c9c21552c915", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, {"status": "released", "experimentset_type": "replicate", "accession": "4DNESSKEUB9C", "@type": ["ExperimentSetReplicate", "ExperimentSet", "Item"], "display_title": "4DNESSKEUB9C", "@id": "/experiment-set-replicates/4DNESSKEUB9C/", "uuid": "2e5d1b70-9dc0-4768-9ebb-decb786f020e", "experiments_in_set": [{"uuid": "48024f95-cfa0-484b-85d6-6e069fd4e8d6", "@type": ["ExperimentMic", "Experiment", "Item"], "display_title": "multiplexed FISH on R1 mESC line - 4DNEXX8WH5HH", "status": "released", "@id": "/experiments-mic/4DNEXX8WH5HH/", "experiment_type": {"@type": ["ExperimentType", "Item"], "status": "released", "@id": "/experiment-types/multiplexed-fish/", "display_title": "multiplexed FISH", "title": "multiplexed FISH", "uuid": "bcdda46a-489d-4d22-be80-c9c21552c915", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, {"status": "released", "experimentset_type": "replicate", "accession": "4DNESWTEQEBQ", "@type": ["ExperimentSetReplicate", "ExperimentSet", "Item"], "display_title": "4DNESWTEQEBQ", "@id": "/experiment-set-replicates/4DNESWTEQEBQ/", "uuid": "10a3171b-6a40-46c9-be5a-22ac86b23221", "experiments_in_set": [{"uuid": "d9e8dc6a-5ac7-46ad-8ae8-cbbd14b87c0b", "@type": ["ExperimentMic", "Experiment", "Item"], "display_title": "multiplexed FISH on R1 mESC line - 4DNEXPY1181B", "status": "released", "@id": "/experiments-mic/4DNEXPY1181B/", "experiment_type": {"@type": ["ExperimentType", "Item"], "status": "released", "@id": "/experiment-types/multiplexed-fish/", "display_title": "multiplexed FISH", "title": "multiplexed FISH", "uuid": "bcdda46a-489d-4d22-be80-c9c21552c915", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "@id": "/publications/0a8a0fdd-eafe-44f3-b7a4-744b50a98b16/", "@type": ["Publication", "Item"], "uuid": "0a8a0fdd-eafe-44f3-b7a4-744b50a98b16", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "Jensen CL et al. 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