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At the lowest resolution, large sections of chromosomes are packed in territories, at a higher level chromosomal regions are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and distal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression. In recent years, a number of methods called chromatin (conformation) capture (CC) have been developed and used to capture dynamic and stable DNA contacts that constitute genome architecture and potential regulatory interactions. A major limitation of these methods is that they all depend on a single crosslinker, formaldehyde, which crosslinks DNA to proteins as well as proteins to other proteins. This complicates the interpretation of the observed 'DNA-DNA' contacts and it lacks distance information. Here we propose an orthogonal strategy, named Distance-Hi-C (D-Hi-C), where we design, test, and apply a battery of photo-activated crosslinkers, designed to directly measure distances between interacting sites genome-wide. These bivalent crosslinkers consist of two reactive groups separated by a linker. DNA intercalaters that can be crosslinked by photo-activation (i.e. Psoralen) will be used as the reactive groups. Linkers will be of varying precise lengths, and either flexible or rigid in nature so that the 3-dimensional distance between crosslinked loci can be inferred. Such DNA-specific bivalent crosslinking reagents, when substituted for formaldehyde in Hi-C protocol, produces space constraints revealing enhancer- promoter interactions and potentially allowing the inference of the 3D arrangement of the nuclear genome with unprecedented precision. Moreover, D-Hi-C is expected to lower backgrounds and allow examination of short and moderate range interactions, which are obscured by high backgrounds of current methods. Addition of groups such as digoxigenin to the bivalent crosslinkers, in addition to biotin incorporated to the ligation products, will enable better purification and thus deeper examination of genomic interactions. Finally, sampling DNA distances in time following gene activation provides a means of exploring the 4D architecture and setting critical limits in evaluating mechanisms of gene activation. The specific aims are to 1) synthesize a battery of bivalent crosslinkers and evaluate their ability to crosslink DNA in vitro; 2) test D-Hi-C crosslinkers relative to formaldehyde in the Drosophila nuclei model; 3) apply new crosslinkers to tier 1, ENCODE GM12878 and K562 cell lines to test their efficacy in human cells; and 4) test locus-specific photo-crosslinking that will allow a more focused and thorough examination of locus-specific interactions with the genome in a time course of gene activation. 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["system.Everyone"], "edit": ["group.admin"]}}, "file_format": {"display_title": "pairs", "uuid": "d13d06cf-218e-4f61-aaf0-91f226248b2c", "status": "released", "file_format": "pairs", "@type": ["FileFormat", "Item"], "@id": "/file-formats/pairs/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "last_modified": {"date_modified": "2024-08-07T22:40:15.918614+00:00"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "track_and_facet_info": {"experimental_lab": "John Lis, CORNELL", "experiment_type": "in situ Hi-C", "experiment_bucket": "HiC Processing Pipeline - v0.3.0", "assay_info": "MboI", "dataset": "HiC on S2 cells +/- Heat Shock", "condition": "Heat Shock", "replicate_info": "Biorep 1, Techrep 1", "biosource_name": "S2", "lab_name": "4DN DCIC, HMS"}}], "title": "HiC Processing Pipeline - v0.3.0", "description": "These are files generated using the updated Hi-C processing pipeline. They should be largely similar to those available in the Processed Files tab, which were generated with the previous version of the standard pipeline.  One potential difference of note is that the version of cooler used to generate the mcool file has a bug fix to prevent a pixel duplication issue which is observed in some files generated by the previous version of the pipeline.  Another notable difference is that a filter is applied to remove reads with MAPQ scores below 30 prior to mcool file generation."}], "biosample_quantity_units": "cells", "crosslinking_temperature": 23.0, "library_preparation_date": "2016-11-21", "fragment_size_selection_method": "see document", "@id": "/experiments-hi-c/4DNEXZEXDG8I/", "@type": ["ExperimentHiC", "Experiment", "Item"], "uuid": "cb94a430-25f6-4d18-af9f-9650b1455734", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "in situ Hi-C on S2 with MboI - 4DNEXZEXDG8I", "external_references": [{"ref": "GEO:GSM3753419", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3753419"}, {"ref": "SRA:SRX5797720", "uri": "https://www.ncbi.nlm.nih.gov/sra/?term=SRX5797720"}], "experiment_sets": [{"status": "released", "@type": ["ExperimentSetReplicate", "ExperimentSet", "Item"], "accession": "4DNESFI64TG3", "uuid": "3b61e4e8-1077-4dcd-b1e7-6634f3fe1988", "@id": "/experiment-set-replicates/4DNESFI64TG3/", "experimentset_type": "replicate", "display_title": "4DNESFI64TG3", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "produced_in_pub": {"short_attribution": "Ray J et al. (2019)", "@id": "/publications/5b2a068c-8de5-4d6f-8ac7-6a274444bfb2/", "date_published": "2019-09-10", "uuid": "5b2a068c-8de5-4d6f-8ac7-6a274444bfb2", "authors": ["Ray J", "Munn PR", "Vihervaara A", "Lewis JJ", "Ozer A", "Danko CG", "Lis JT"], "abstract": "Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes  in transcription that are accompanied by chromatin decondensation and nucleosome  loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress  affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.", "title": "Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock.", "ID": "PMID:31506350", "@type": ["Publication", "Item"], "status": "current", "display_title": "Ray J et al. 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Here we have employed in situ Hi-C to determine how heat stress  affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.", "status": "current", "journal": "Proceedings of the National Academy of Sciences of the United States of America", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}], "experiment_categorizer": {"field": "Enzyme", "value": "MboI", "combined": "Enzyme: MboI"}, "experiment_summary": "in situ Hi-C on S2 with MboI", "@context": "/terms/", "aggregated-items": {"badges": [{"parent": "/biosamples/4DNBSQL84T59/", "embedded_path": "biosample.badges", "item": {"messages": ["Biosample missing doubling_number", "Biosample missing passage_number", "Biosample missing culture_duration", "Biosample missing morphology_image"], "badge": {"commendation": null, "warning": "Biosample Metadata Incomplete", "uuid": "2b2cc7ff-b7a8-4138-9a6c-22884fc71690", "@id": "/badges/biosample-metadata-incomplete/", "badge_icon": "/static/img/badges/biosample-icon.svg", "description": "Biosample is missing metadata information required as part of the standards implemented by the 4DN Samples working group."}}}]}, "validation-errors": []}