{"ID": "doi:10.1101/2023.02.06.527380", "lab": {"title": "Alistair Boettiger, STANFORD", "display_title": "Alistair Boettiger, STANFORD", "uuid": "312cb909-76a6-405d-a96c-c3292abf08a1", "@type": ["Lab", "Item"], "correspondence": [{"contact_email": "YWJvZXR0aWdAc3RhbmZvcmQuZWR1", "@id": "/users/b8835c78-05e3-4173-a6c5-1ab93b4d12cc/", "display_title": "Alistair Boettiger"}], "@id": "/labs/alistair-boettiger-lab/", "status": "current", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.312cb909-76a6-405d-a96c-c3292abf08a1"]}}, "url": "http://biorxiv.org/lookup/doi/10.1101/2023.02.06.527380", "award": {"name": "1U01DK127419-01", "uuid": "b7c5d0c8-053e-4da3-b446-b68787a5a738", "center_title": "Bintu", "project": "4DN", "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", "display_title": "LIVE-CELL MULTIPLEX SUPER-RESOLUTION IMAGING OF CHROMATIN STATE TRANSITIONS", "@id": "/awards/1U01DK127419-01/", "@type": ["Award", "Item"], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "title": "Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development", "status": "replaced", "aliases": ["4dn-dcic-lab:hung_et_al_biorxiv_limbbud"], "authors": ["Hung T-C", "Kingsley DM", "Boettiger AN"], "journal": "bioRxiv", "version": "1", "abstract": "While long-range enhancers and their target promoters are frequently contained within a TAD, many developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling such cross-TAD interactions remain to be assessed. To test these hypotheses, we use Optical Reconstruction of Chromatin Architecture (ORCA) to characterize the conformations of the Pitx1 locus on thousands of single chromosomes in developing mouse limbs. Our data supports a model in which neighboring boundaries are stacked with each other as a result of loop-extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also explains the appearance of architectural stripes in the population average maps (e.g. Hi-C data). Through molecular dynamics simulations, we further propose that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders function, both facilitating as well as preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.", "date_created": "2023-02-08T14:19:38.633834+00:00", "published_by": "4DN", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2024-01-22T19:45:31.599169+00:00"}, "date_published": "2023-02-06", "public_release": "2023-02-08", "schema_version": "2", "static_content": [{"content": {"lab": {"@id": "/labs/alistair-boettiger-lab/", "status": "current", "display_title": "Alistair Boettiger, STANFORD", "uuid": "312cb909-76a6-405d-a96c-c3292abf08a1", "@type": ["Lab", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.312cb909-76a6-405d-a96c-c3292abf08a1"]}}, "@type": ["StaticSection", "UserContent", "Item"], "content_as_html": "<div class=\"markdown-container\"><p>This biorxiv set was replaced by <a href=\"https://data.4dnucleome.org/3a910b1f-5fd7-4684-b2a5-e6f2baaecceb/\" rel=\"noopener noreferrer\" target=\"_blank\">PMID:38238628</a>.</p></div>", "@id": "/static-sections/ce0d15d1-e0c8-4e48-bca0-6deb5599ebf7/", "status": "released", "content": "This biorxiv set was replaced by [PMID:38238628](https://data.4dnucleome.org/3a910b1f-5fd7-4684-b2a5-e6f2baaecceb/).", "filetype": "md", "uuid": "ce0d15d1-e0c8-4e48-bca0-6deb5599ebf7", "display_title": "Note: Replaced Biorxiv", "options": {"filetype": "md", "title_icon": "info", "collapsible": false, "default_open": true, "convert_ext_links": true}, "name": "static-header.replaced_item_5c5491a8-279d-4850-940d-e62af0223f52", "award": {"@type": ["Award", "Item"], "@id": "/awards/1U01DK127419-01/", "uuid": "b7c5d0c8-053e-4da3-b446-b68787a5a738", "display_title": "LIVE-CELL MULTIPLEX SUPER-RESOLUTION IMAGING OF CHROMATIN STATE TRANSITIONS", "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "title": "Note: Replaced Biorxiv", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.owner", "userid.7677f8a8-79d2-4cff-ab0a-a967a2a68e39"]}}, "location": "header"}], "project_release": "2023-02-08", "exp_sets_prod_in_pub": [{"experimentset_type": "replicate", "display_title": "4DNES4TC13IL", "@type": ["ExperimentSetReplicate", "ExperimentSet", "Item"], "uuid": "47229cdb-9e28-413b-918c-3bb45a444cb0", "status": "released", "@id": "/experiment-set-replicates/4DNES4TC13IL/", "accession": "4DNES4TC13IL", "experiments_in_set": [{"@type": ["ExperimentMic", "Experiment", "Item"], "@id": "/experiments-mic/4DNEXXACPQZA/", "display_title": "multiplexed FISH on limb bud - 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