{"lab": {"uuid": "bd4a15cf-e3f4-4938-933e-787883a48d7c", "title": "Danwei Huangfu, MSKCC", "correspondence": [{"contact_email": "aHVhbmdmdWRAbXNrY2Mub3Jn", "@id": "/users/b9ddcafc-fc2d-401d-98a6-dd1cb39b2982/", "display_title": "Danwei Huangfu"}], "display_title": "Danwei Huangfu, MSKCC", "status": "current", "@type": ["Lab", "Item"], "@id": "/labs/danwei-huangfu-lab/", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.bd4a15cf-e3f4-4938-933e-787883a48d7c"]}}, "award": {"name": "1U01DK128852-01", "@id": "/awards/1U01DK128852-01/", "uuid": "8432a44c-4da7-446b-9080-f4fae299ebd6", "center_title": "OFHHD - Huangfu", "status": "current", "project": "4DN", "display_title": "DISCOVERY OF DIABETES-RELEVANT BETA CELL ENHANCERS THROUGH 4D ENHANCER MAPPING, INTEGRATIVE ANALYSIS, AND LARGE-SCALE CRISPRI PERTURBATION SCREENS", "description": "OFHHD: Enhancers are essential regulatory elements that together with transcription factors (TFs) instruct cell- type specific transcriptional programs during development, tissue homeostasis and regeneration. Initiatives such as the ENCODE project, revealed tens of thousands putative enhancers based on linear proximity, using criteria like chromatin accessibility, TF binding, and histone modifications such as H3K27ac. However, a main challenge of uncovering functional enhancers and assigning them to target genes lies in the complexity of the 3D chromatin organization, which can influence enhancer specificity and activity. Using an advanced chromosome conformation capture assay, we recently captured the dynamic rewiring of 3D enhancer networks during mouse somatic cell reprogramming and discovered multi-connected enhancers that we named \u201c3D enhancer hubs\u201d. Here we extend the 3D mapping approach to human primary islets, and compare islets from healthy and type 2 diabetes (T2D) donors to assemble a 4D atlas to capture the rewiring of 3D enhancer network in disease progression. At the same time, we plan to compare the enhancer network in adult islets to earlier stages of development by using human pluripotent stem cells (hPSCs) to generate early \u03b2 cells and their developmental precursors. Utilizing these 4D genomic data, we will computationally nominate core \u03b2-cell specific enhancers relevant to \u03b2 cell development, function, and T2D, and then interrogate these putative enhancers through large-scale CRISPRi mediated perturbation screens using hPSC-\u03b2 cells. Enhancers identified from the screening effort will be further validated in an established human \u03b2 cell line and primary human islet \u03b2 cells. This proposal addresses a critical gap in the 4DN initiative, that is how to translate 3D genomics data into functional data with respect to gene expression in the context of human health. Successful completion of our aims will establish a paradigm for the discovery and interrogation of functional enhancers that instruct transcriptional programs specific to a cell type of interest, reveal unique insights into their mechanisms of action, and identify enhancers with relevance to human development and disease. For instance, uncovering functional enhancers could assist the identification of noncoding causal variants identified in genome-wide association studies.", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "pi": {"error": "no view permissions"}}, "study": "Early pancreatic differentiation", "badges": [{"badge": {"warning": "Replicate Numbers", "@type": ["Badge", "Item"], "@id": "/badges/replicate-numbers/", "badge_icon": "/static/img/badges/replicates-orange-circle.svg", "description": "Issues with replicate numbers", "badge_classification": "Warning", "display_title": "Replicate Numbers", "uuid": "24a64a84-3c33-4d76-aaf2-e5ef45eff347", "status": "released", "title": "Replicate Numbers", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "messages": ["Replicate set contains only a single biological replicate"]}], "status": "released", "aliases": ["danwei-huang-lab:hi-c_sox17-eGFP_idCAS9-KRAB_HUES8_undiff"], "accession": "4DNESQMUTYXH", "condition": "undifferentiated - HUES8", "description": "in situ Hi-C on undifferentiated SOX17-eGFP idCAS9-KRAB HUES8 human embryonic stem cells", "study_group": "Time Course", "date_created": "2023-05-31T15:11:34.562013+00:00", "submitted_by": {"error": "no view permissions"}, "dataset_label": "in situ Hi-C on hESCs differentiated toward pancreatic cells", "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2024-07-31T14:45:48.204729+00:00"}, "public_release": "2023-06-09", "replicate_exps": [{"bio_rep_no": 1, "tec_rep_no": 1, "replicate_exp": {"accession": "4DNEXTXPUCC1", "status": "released", "@type": ["ExperimentHiC", "Experiment", "Item"], "display_title": "in situ Hi-C on SOX17-eGFP idCAS9-KRAB HUES8 with Arima - A1, A2 - 4DNEXTXPUCC1", "uuid": "e43c8913-5897-455b-be2d-bcd777dd7c8f", "@id": "/experiments-hi-c/4DNEXTXPUCC1/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}}], "schema_version": "2", "static_content": [{"content": {"title": "4DNESQMUTYXH - Processed files", "description": "4DNESQMUTYXH (in situ Hi-C on undifferentiated SOX17-eGFP idCAS9-KRAB HUES8 human embryonic stem cells): 4DNFI1YXTFOH, 4DNFIJC7POCX, 4DNFIEOGSLA4, 4DNFIFW6LCWK", "@id": "/higlass-view-configs/5cf8967c-0b14-4a1a-aba1-32cd0e7bd249/", "name": "5cf8967c-0b14-4a1a-aba1-32cd0e7bd249", "contributing_labs": [{"display_title": "Christina Leslie, MSKCC", "@type": ["Lab", "Item"], "@id": "/labs/christina-leslie-lab/", "status": "current", "uuid": "b533abb2-66ea-4f89-b34f-22ba1954b38a", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b533abb2-66ea-4f89-b34f-22ba1954b38a"]}}, {"display_title": "Effie Apostolou, CORNELL", "@type": ["Lab", "Item"], "@id": "/labs/effie-apostolou-lab/", "status": "current", "uuid": "25604a5f-772b-43e2-a004-136ce41d0090", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.25604a5f-772b-43e2-a004-136ce41d0090"]}}], "award": {"uuid": "8432a44c-4da7-446b-9080-f4fae299ebd6", "@type": ["Award", "Item"], "@id": "/awards/1U01DK128852-01/", "status": "current", "display_title": "DISCOVERY OF DIABETES-RELEVANT BETA CELL ENHANCERS THROUGH 4D ENHANCER MAPPING, INTEGRATIVE ANALYSIS, AND LARGE-SCALE CRISPRI PERTURBATION SCREENS", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "lab": {"display_title": "Danwei Huangfu, MSKCC", "@type": ["Lab", "Item"], "@id": "/labs/danwei-huangfu-lab/", "uuid": "bd4a15cf-e3f4-4938-933e-787883a48d7c", "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.bd4a15cf-e3f4-4938-933e-787883a48d7c"]}}, "status": "released", "filetype": "HiglassViewConfig", "uuid": "5cf8967c-0b14-4a1a-aba1-32cd0e7bd249", "display_title": "4DNESQMUTYXH - Processed files", "@type": ["HiglassViewConfig", "UserContent", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.owner", "userid.7677f8a8-79d2-4cff-ab0a-a967a2a68e39"]}}, "location": "tab:processed-files", "description": "auto_generated_higlass_view_config"}], "static_headers": [{"lab": {"@id": "/labs/4dn-dcic-lab/", "@type": ["Lab", "Item"], "status": "current", "display_title": "4DN DCIC, HMS", "uuid": "828cd4fe-ebb0-4b36-a94a-d2e3a36cc989", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.828cd4fe-ebb0-4b36-a94a-d2e3a36cc989"]}}, "body": "<b>In Situ Hi-C</b>\n\n<p>\n   In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. 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. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n</p>\n<p>\nSee <a href=\"https://www.sciencedirect.com/science/article/pii/S0092867414014974?via%3Dihub\">Rao et al., 2014</a> for more details.\n</p>\n\n<div>\n<img style=\"width: 600px;max-width:100%;\" src=\"https://s3.amazonaws.com/4dn-dcic-public/static-pages/InfoBoxes/IsHC_fig1.png\"/>\n  <br/><br/>\n  <em>Image source: Rao et al., 2014, Figure 1A</em>\n</div>", "filetype": "html", "content_as_html": "<div class=\"html-container\"><b>In Situ Hi-C</b>\n<p>\n   In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. 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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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."], "file_size": 928215072, "uuid": "3f95c326-9baf-417b-8e1c-6132b140e98d", "href": "/files-processed/4DNFI1YXTFOH/@@download/4DNFI1YXTFOH.mcool", "@id": "/files-processed/4DNFI1YXTFOH/", "file_type": "contact matrix", "upload_key": "3f95c326-9baf-417b-8e1c-6132b140e98d/4DNFI1YXTFOH.mcool", "file_type_detailed": "contact matrix (mcool)", "genome_assembly": "GRCh38", "higlass_uid": "74b2464e-9f37-431e-b94f-048b07b9d6ff", "file_format": {"display_title": "mcool", "@id": "/file-formats/mcool/", "@type": ["FileFormat", "Item"], "uuid": "d13d06cf-218e-4f61-ccf0-91f226248b2c", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "open_data_url": "https://4dn-open-data-public.s3.amazonaws.com/fourfront-webprod/wfoutput/3f95c326-9baf-417b-8e1c-6132b140e98d/4DNFI1YXTFOH.mcool", "display_title": "4DNFI1YXTFOH.mcool", 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Our gene regulatory network modeling identified that nonlinear  enhancer gene regulation during cell state transitions can be leveraged to  improve the sensitivity of enhancer discovery. Using human embryonic stem cell  definitive endoderm differentiation as a dynamic transition system, we conducted  a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set  of enhancers for each of the core endoderm-specifying transcription factors. Many  enhancers had strong effects mid-transition but weak effects post-transition,  consistent with the nonlinear temporal responses to enhancer perturbation  predicted by the modeling. Integrating three-dimensional genomic information, we  were able to develop a CTCF-loop-constrained Interaction Activity model that can  better predict functional enhancers compared to models that rely on Hi-C-based  enhancer-promoter contact frequency. Our study provides generalizable strategies  for sensitive and systematic enhancer discovery in both normal and pathological  cell state transitions.", "display_title": "Luo R et al. (2023) PMID:37488417", "journal": "Nature genetics", "authors": ["Luo R", "Yan J", "Oh JW", "Xi W", "Shigaki D", "Wong W", "Cho HS", "Murphy D", "Cutler R", "Rosen BP", "Pulecio J", "Yang D", "Glenn RA", "Chen T", "Li QV", "Vierbuchen T", "Sidoli S", "Apostolou E", "Huangfu D", "Beer MA"], "title": "Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear  enhancer gene regulatory activity during cell state transitions.", "date_published": "2023-08", "@type": ["Publication", "Item"], "short_attribution": "Luo R et al. 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Through multiple CRISPR-activation screens, we discovered human ESCs have  pre-established transcriptionally competent chromatin regions (CCRs) that support  lineage-specific gene expression at levels comparable to differentiated cells.  CCRs reside in the same topological domains as their target genes. They lack  typical enhancer-associated histone modifications but show enriched occupancy of  pluripotent transcription factors, DNA demethylation factors, and histone  deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while  HDAC1 family members prevent premature activation. This \"push and pull\" feature  resembles bivalent domains at developmental gene promoters but involves distinct  molecular mechanisms. Our study provides new insights into pluripotency  regulation and cellular plasticity in development and disease. ONE SENTENCE  SUMMARY: We report a class of distal regulatory regions distinct from enhancers  that confer human embryonic stem cells with the competence to rapidly activate  the expression of lineage-specific genes.", "journal": "bioRxiv : the preprint server for biology", "date_published": "2023-06-14", "status": "current", "display_title": "Pulecio J et al. 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