{"lab": {"display_title": "Gerd Blobel, CHOP", "status": "current", "correspondence": [{"contact_email": "YmxvYmVsQGVtYWlsLmNob3AuZWR1", "@id": "/users/9dcbdd0c-634c-4ec6-b6dc-89efd542c203/", "display_title": "Gerd Blobel"}], "@id": "/labs/gerd-blobel-lab/", "title": "Gerd Blobel, CHOP", "uuid": "00073229-0955-47af-ab67-6abf4880cb2c", "@type": ["Lab", "Item"], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.00073229-0955-47af-ab67-6abf4880cb2c"]}}, "award": {"project": "4DN", "center_title": "NT - Raj", "description": "NT: Critical unanswered questions in the field of genome biology are how the dynamics of chromatin folding shape gene expression patterns. Our knowledge of the dynamics of higher-order 3-D folding of chromatin is severely limited, largely due to the lack of technologies to precisely image, engineer and monitor looping in a precise spatiotemporal manner across a population of cells. Here we propose to address these limitations by developing tools to dynamically alter chromatin folding in a synchronous manner across populations of cells as well as individual cells, and measure chromatin looping and its relationship to transcription at high spatial resolution in single cells. In Specific Aim 1 we will design tools to control looping dynamics. We will modify factors that fold chromatin at various levels, such as Ldb1 and CTCF by fusion to a moiety whose stability can be controlled by diffusible ligands. In combination with hi resolution 5C and single molecule imaging these tools are expected to generate fundamental insights into the relationship of nuclear architecture and gene expression mechanisms. In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develp reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. 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It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed <i>in situ</i> inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n</p>\n<p>\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. 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It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed <i>in situ</i> inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n</p>\n<p>\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. 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Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed <i>in situ</i> inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n</p>\n<p>\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. 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This duplication does not affect the higlass display of these files, howevver, downstream analyses using this file may encounter issues due to this pixel duplication.  The counts from the duplicate pixels can be aggregated to determine the correct count value at that location. If this issue is problematic for your needs you should consider regenerating the matrices from the merged pairs file of the associated dataset using a more recent version of cooler.  We are working to update the pipeline but do not yet have a predicted date for when this issue will be resolved."], "href": "/files-processed/4DNFIRAHQIGN/@@download/4DNFIRAHQIGN.mcool", "md5sum": "7b06563d88adaa930939aa2692167d15", "higlass_uid": "8d4ffc67-3197-4a28-a197-b76afd5e7937", "file_classification": "processed file", "file_format": {"@type": ["FileFormat", "Item"], "display_title": "mcool", "uuid": "d13d06cf-218e-4f61-ccf0-91f226248b2c", "@id": "/file-formats/mcool/", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "accession": "4DNFIRAHQIGN", "file_size": 2346137204, "uuid": "77435de5-d768-4fae-9867-4dbc03f82f92", "display_title": "4DNFIRAHQIGN.mcool", "status": "released", "file_type_detailed": "contact matrix (mcool)", "@id": "/files-processed/4DNFIRAHQIGN/", "@type": ["FileProcessed", "File", "Item"], "open_data_url": 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Contact domains form from the 'bottom up'-smaller  subTADs are formed initially, followed by convergence into multi-domain TAD structures. CTCF is partially retained on mitotic chromosomes and immediately resumes full binding in ana/telophase. By contrast, cohesin is completely evicted from mitotic chromosomes and regains focal binding at a slower rate. The formation of CTCF/cohesin co-anchored structural loops follows the kinetics of cohesin positioning. Stripe-shaped contact patterns-anchored by CTCF-grow in length, which is consistent with a loop-extrusion process after mitosis. Interactions between cis-regulatory elements can form rapidly, with rates exceeding those of CTCF/cohesin-anchored contacts. Notably, we identified a group of rapidly emerging transient contacts between cis-regulatory elements in ana/telophase that are dissolved upon G1 entry, co-incident with the establishment of inner boundaries or nearby interfering chromatin loops. We also  describe the relationship between transcription reactivation and architectural features. Our findings indicate that distinct but mutually influential forces drive post-mitotic chromatin reconfiguration.", "@type": ["Publication", "Item"], "title": "Chromatin structure dynamics during the mitosis-to-G1 phase transition.", "ID": "PMID:31776509", "status": "current", "journal": "Nature", "uuid": "94eaa29d-46e7-420c-ad92-a7343847020a", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "pubs_using": [], "publications_of_set": [{"display_title": "Zhang H et al. (2019) PMID:31776509", "date_published": "2019-12", "abstract": "Features of higher-order chromatin organization-such as A/B compartments, topologically associating domains and chromatin loops-are temporarily disrupted during mitosis(1,2). Because these structures are thought to influence gene regulation, it is important to understand how they are re-established after mitosis. Here we examine the dynamics of chromosome reorganization by Hi-C after  mitosis in highly purified, synchronous mouse erythroid cell populations. We observed rapid establishment of A/B compartments, followed by their gradual intensification and expansion. Contact domains form from the 'bottom up'-smaller  subTADs are formed initially, followed by convergence into multi-domain TAD structures. CTCF is partially retained on mitotic chromosomes and immediately resumes full binding in ana/telophase. By contrast, cohesin is completely evicted from mitotic chromosomes and regains focal binding at a slower rate. The formation of CTCF/cohesin co-anchored structural loops follows the kinetics of cohesin positioning. Stripe-shaped contact patterns-anchored by CTCF-grow in length, which is consistent with a loop-extrusion process after mitosis. Interactions between cis-regulatory elements can form rapidly, with rates exceeding those of CTCF/cohesin-anchored contacts. Notably, we identified a group of rapidly emerging transient contacts between cis-regulatory elements in ana/telophase that are dissolved upon G1 entry, co-incident with the establishment of inner boundaries or nearby interfering chromatin loops. We also  describe the relationship between transcription reactivation and architectural features. 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