{"ID": "doi:10.1083/jcb.201807108", "lab": {"title": "Andrew Belmont, ILLINOIS", "uuid": "b2c2deeb-e883-4ac0-b9e2-906e598884d6", "@type": ["Lab", "Item"], "display_title": "Andrew Belmont, ILLINOIS", "@id": "/labs/andrew-belmont-lab/", "correspondence": [{"contact_email": "YXNiZWxAaWxsaW5vaXMuZWR1", "@id": "/users/92f90aed-7df1-4bd9-9e74-a472cb50d663/", "display_title": "Andrew Belmont"}], "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b2c2deeb-e883-4ac0-b9e2-906e598884d6"]}, "pi": {"error": "no view permissions"}}, "url": "https://www.ncbi.nlm.nih.gov/pubmed/30154186", "award": {"status": "current", "display_title": "COMBINED CYTOLOGICAL, GENOMIC, AND FUNCTIONAL MAPPING OF NUCLEAR GENOME ORGANIZATION", "description": "NOFIC: Decades of microscopy have revealed that the nucleus is not a homogeneous organelle, but rather consists of distinct compartments such as nucleoli, nuclear speckles, the nuclear lamina, among other structures. Increasing evidence indicates that specific genomic regions each associate with these compartments. This genome compartmentalization has been linked to various functions, but these links are still poorly understood. Interestingly, Lamina Associated Domains (LADs) share specific heterochromatin marks, defining chromatin domains with distinct genetic and epigenetic properties. Genomic regions associating with other nuclear compartments may similarly define distinct classes of chromatin domains. One major bottleneck towards a deeper understanding of nuclear organization has been the inability to convert microscopy views of nuclear compartments into genome-wide maps that show which loci are associated with which compartment, and how the chromosomal fiber traverses between compartments. In addition, there is an urgent need for more efficient methods to dissect the mechanisms by which large genomic regions are targeted to specific nuclear compartments. Finally, there is an urgent need for high-throughput approaches that query the functional relevance of genome compartmentalization. For this Center grant, we propose to meet these needs through the following Aims: 1. Develop a strategy that connects microscopy views to genome-wide maps that, together with modeling, reveal the localization and dynamics of genomic regions relative to all major nuclear compartments. 2. Develop methods for efficient manipulation of the genome in order to elucidate mechanisms that target loci to specific compartments. 3. Develop methods to measure, model, and validate the functional relevance of nuclear compartments. The combined results of these approaches will reveal causal relationships now hidden among entangled genomic, epigenetic, and nuclear organization features. Deliverables of this proposal include a wide range of structural and functional maps of nuclear organization, reagents for visualizing endogenous chromosome loci, a powerful pipeline for synthesis of ~100kb DNA fragments, and cell lines facilitating repeated, high-fidelity insertio of these large fragments back into selected sites in the genome. These resources will provide a powerful complement to other 4D Nucleome Consortium efforts. A key strength of this Center proposal is the experience and complementary research capabilities of its five Investigators. Together they will pool their expertise for a concerted investigation into the biological functions of nuclear compartmentalization.", "center_title": "NOFIC - Belmont", "name": "1U54DK107965-01", "project": "4DN", "uuid": "91b694c3-f4d7-4ddd-8278-16f94e15c1c5", "@type": ["Award", "Item"], "@id": "/awards/1U54DK107965-01/", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "title": "Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler.", "status": "current", "aliases": ["belmont-lab:tsa-seq-1-in-k562-JCB"], "authors": ["Chen Y", "Zhang Y", "Wang Y", "Zhang L", "Brinkman EK", "Adam SA", "Goldman R", "van Steensel B", "Ma J", "Belmont AS"], "journal": "The Journal of cell biology", "abstract": "While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a \"cytological ruler\" for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription \"hot zones.\" Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression.", "categories": ["technology development", "basic biology"], "date_created": "2018-12-06T20:03:38.904708+00:00", "published_by": "4DN", "submitted_by": {"error": "no view permissions"}, "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2019-11-12T20:35:03.632825+00:00"}, "date_published": "2018-11-05", "public_release": "2019-04-09", "schema_version": "2", "static_content": [{"content": {"title": "About TSA-seq", "status": "released", "lab": {"@id": "/labs/andrew-belmont-lab/", "uuid": "b2c2deeb-e883-4ac0-b9e2-906e598884d6", "display_title": "Andrew Belmont, ILLINOIS", "status": "current", "@type": ["Lab", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b2c2deeb-e883-4ac0-b9e2-906e598884d6"]}}, "@id": "/static-sections/802d2154-e8d1-4c48-bd1d-42b3d92bd5c5/", "@type": ["StaticSection", "UserContent", "Item"], "display_title": "About TSA-seq", "uuid": "802d2154-e8d1-4c48-bd1d-42b3d92bd5c5", "content": "

TSA sequencing (TSA-Seq), is a genomic method that allows the estimation of cytological distances of chromosome loci genome-wide relative to a particular nuclear compartment and can be used to infer chromosome trajectories from one compartment to another.

\n

TSA uses an antibody-coupled HRP in conjunction with an antibody targeting a specific protein or compartment to catalyze the formation of diffusible biotin-tyramide free radicals and create a free radical concentration gradient centered at the target in fixed nuclei. The TSA reaction is followed by the reversal of formaldehyde cross-linking, DNA isolation, pulldown of biotinylated DNA, and high-throughput sequencing. TSA labels DNA directly and the steady-state concentration of tyramide free radicals during the TSA reaction can be modeled by a simple exponential decay proportional to the distance from the target.

\n

TSA enrichment genomic maps are generated by plotting the log2 ratio of the normalized pulldown read density versus the normalized read density of input DNA over a 20-kbp sliding window and can be visualized as normalized counts in 1D bigwig tracks.

", "award": {"@id": "/awards/1U01CA200059-01/", "uuid": "b0b9c607-f8b4-4f02-93f4-9895b461334b", "status": "current", "display_title": "4D NUCLEOME NETWORK DATA COORDINATION AND INTEGRATION CENTER - PHASE I", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "content_as_html": "

TSA sequencing (TSA-Seq), is a genomic method that allows the estimation of cytological distances of chromosome loci genome-wide relative to a particular nuclear compartment and can be used to infer chromosome trajectories from one compartment to another.

\n

TSA uses an antibody-coupled HRP in conjunction with an antibody targeting a specific protein or compartment to catalyze the formation of diffusible biotin-tyramide free radicals and create a free radical concentration gradient centered at the target in fixed nuclei. The TSA reaction is followed by the reversal of formaldehyde cross-linking, DNA isolation, pulldown of biotinylated DNA, and high-throughput sequencing. TSA labels DNA directly and the steady-state concentration of tyramide free radicals during the TSA reaction can be modeled by a simple exponential decay proportional to the distance from the target.

\n

TSA enrichment genomic maps are generated by plotting the log2 ratio of the normalized pulldown read density versus the normalized read density of input DNA over a 20-kbp sliding window and can be visualized as normalized counts in 1D bigwig tracks.

", "name": "chen-etal.tsa-seq-technique-summary", "options": {"filetype": "html", "collapsible": false, "default_open": true}, "filetype": "html", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.owner", "userid.986b362f-4eb6-4a9c-8173-3ab267227777"]}}, "location": "tab:overview"}, {"content": {"title": "About this study", "status": "released", "lab": {"@id": "/labs/andrew-belmont-lab/", "uuid": "b2c2deeb-e883-4ac0-b9e2-906e598884d6", "display_title": "Andrew Belmont, ILLINOIS", "status": "current", "@type": ["Lab", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b2c2deeb-e883-4ac0-b9e2-906e598884d6"]}}, "@id": "/static-sections/e5adca2f-f3b9-4e20-8ef4-aa220d63a449/", "@type": ["StaticSection", "UserContent", "Item"], "display_title": "About this study", "uuid": "e5adca2f-f3b9-4e20-8ef4-aa220d63a449", "content": "

This publication describes the technology development of the TSA-seq method and it's application in K562 cells to examine the distribution of genome loci relative to nuclear speckles and nuclear lamina and well as proximity to markers of active domains of transcription.

\n

The publication discusses the relationship of previously identified chromatin compartments and TSA-seq signal and distance estimations. Several datasets using this TSA-seq version 1 method were produced for this publication and are shown in the Experiments Summary tab.

\n

In addition Supplementary Table 4 from this publication which is an excel workbook that lists external datasets used in this analysis as confirmative and comparative data is available at this link. NOTE: The analysis presented in this paper and the files referenced in this table are done on human assembly hg19 while the processed files available on the 4DN data portal have been reanalyzed on the hg38 assembly.

", "award": {"@id": "/awards/1U01CA200059-01/", "uuid": "b0b9c607-f8b4-4f02-93f4-9895b461334b", "status": "current", "display_title": "4D NUCLEOME NETWORK DATA COORDINATION AND INTEGRATION CENTER - PHASE I", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "content_as_html": "

This publication describes the technology development of the TSA-seq method and it's application in K562 cells to examine the distribution of genome loci relative to nuclear speckles and nuclear lamina and well as proximity to markers of active domains of transcription.

\n

The publication discusses the relationship of previously identified chromatin compartments and TSA-seq signal and distance estimations. Several datasets using this TSA-seq version 1 method were produced for this publication and are shown in the Experiments Summary tab.

\n

In addition Supplementary Table 4 from this publication which is an excel workbook that lists external datasets used in this analysis as confirmative and comparative data is available at this link. NOTE: The analysis presented in this paper and the files referenced in this table are done on human assembly hg19 while the processed files available on the 4DN data portal have been reanalyzed on the hg38 assembly.

", "name": "hen-etal.paper-summary", "options": {"filetype": "html", "collapsible": false, "default_open": true}, "filetype": "html", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.owner", "userid.986b362f-4eb6-4a9c-8173-3ab267227777"]}}, "location": "tab:overview"}, {"content": {"title": "Summary of Data Sets in this Publication", "status": "released", "lab": {"@id": "/labs/andrew-belmont-lab/", "uuid": "b2c2deeb-e883-4ac0-b9e2-906e598884d6", "display_title": "Andrew Belmont, ILLINOIS", "status": "current", "@type": ["Lab", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b2c2deeb-e883-4ac0-b9e2-906e598884d6"]}}, "@id": "/static-sections/b3518b22-1e71-4625-89ed-bf18c67cc7dc/", "@type": ["StaticSection", "UserContent", "Item"], "display_title": "Summary of Data Sets in this Publication", "uuid": "b3518b22-1e71-4625-89ed-bf18c67cc7dc", "content": "\n\n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n
TargetReaction ConditionInputTSA reactionBio
Reps		Processed Files
SON - as a specific marker for nuclear speckles
No primary antibody controlStandard Buffer (condition 1)4DNESOSG1ECS4DNES67IYWBB1Per replicate Normalized counts
anti-SON HPA023535Standard Buffer (condition 1)4DNES2UMO8MC4DNESSVB91CQ3Per replicate Normalized counts and Predicted distances for replicate 1
50% Glucose (condition 2)4DNESMV75X4B4DNESS84SF2T2Per replicate Normalized counts and Predicted distances for replicate 1
50% Glucose + 0.1 mM DTT (condition 3)4DNESEFCYCWK4DNESTIK63391Per replicate Normalized counts and Predicted distances for replicate 1
anti-SON Belmont lab50% Glucose (condition 2)4DNES27KAIML4DNESYJEVPEW1Per replicate Normalized counts
phosphorylated SC35 - as an independent marker for nuclear speckles
No primary antibody controlStandard Buffer (condition 1)4DNES5C2KRSP4DNESNZGPYE61Per replicate Normalized counts
anti-phophoSC35 S4045Standard Buffer (condition 1)4DNESYP9HV8H4DNESNER8V111Per replicate Normalized counts
Lamin A/C - as a marker for nuclear lamina
No primary antibody controlStandard Buffer (condition 1)4DNESD1KO9LC4DNESQBBEUUS1Per replicate Normalized counts
50% Glucose (condition 2)4DNES1SV4Q4D4DNESVZYLFAN1Per replicate Normalized counts
anti-LaminA/C P0254550% Glucose (condition 2)4DNESX7F2Q3N4DNES19URNAH2Per replicate Normalized counts
Lamin B1 - as a marker for nuclear lamina
No primary antibody control50% Glucose (condition 2)4DNES3IB82SC4DNESWB193842Per replicate Normalized counts
anti-LaminB50% Glucose (condition 2)4DNES55KQ5VP4DNESA1Z2ZVR2Per replicate Normalized counts
Pol2A/RPB1 - as a marker for active regions
No primary antibody controlStandard Buffer (condition 1)4DNESVTOWO4Z4DNES3HEGJ6H1Per replicate Normalized counts
anti-Pol2A 05-623Standard Buffer (condition 1)4DNES1QS8EMG4DNESURW4RWH2Per replicate Normalized counts
\n", "award": {"@id": "/awards/1U01CA200059-01/", "uuid": "b0b9c607-f8b4-4f02-93f4-9895b461334b", "status": "current", "display_title": "4D NUCLEOME NETWORK DATA COORDINATION AND INTEGRATION CENTER - PHASE I", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "content_as_html": "
\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
TargetReaction ConditionInputTSA reactionBio
Reps		Processed Files
SON - as a specific marker for nuclear speckles
No primary antibody controlStandard Buffer (condition 1)4DNESOSG1ECS4DNES67IYWBB1Per replicate Normalized counts
anti-SON HPA023535Standard Buffer (condition 1)4DNES2UMO8MC4DNESSVB91CQ3Per replicate Normalized counts and Predicted distances for replicate 1
50% Glucose (condition 2)4DNESMV75X4B4DNESS84SF2T2Per replicate Normalized counts and Predicted distances for replicate 1
50% Glucose + 0.1 mM DTT (condition 3)4DNESEFCYCWK4DNESTIK63391Per replicate Normalized counts and Predicted distances for replicate 1
anti-SON Belmont lab50% Glucose (condition 2)4DNES27KAIML4DNESYJEVPEW1Per replicate Normalized counts
phosphorylated SC35 - as an independent marker for nuclear speckles
No primary antibody controlStandard Buffer (condition 1)4DNES5C2KRSP4DNESNZGPYE61Per replicate Normalized counts
anti-phophoSC35 S4045Standard Buffer (condition 1)4DNESYP9HV8H4DNESNER8V111Per replicate Normalized counts
Lamin A/C - as a marker for nuclear lamina
No primary antibody controlStandard Buffer (condition 1)4DNESD1KO9LC4DNESQBBEUUS1Per replicate Normalized counts
50% Glucose (condition 2)4DNES1SV4Q4D4DNESVZYLFAN1Per replicate Normalized counts
anti-LaminA/C P0254550% Glucose (condition 2)4DNESX7F2Q3N4DNES19URNAH2Per replicate Normalized counts
Lamin B1 - as a marker for nuclear lamina
No primary antibody control50% Glucose (condition 2)4DNES3IB82SC4DNESWB193842Per replicate Normalized counts
anti-LaminB50% Glucose (condition 2)4DNES55KQ5VP4DNESA1Z2ZVR2Per replicate Normalized counts
Pol2A/RPB1 - as a marker for active regions
No primary antibody controlStandard Buffer (condition 1)4DNESVTOWO4Z4DNES3HEGJ6H1Per replicate Normalized counts
anti-Pol2A 05-623Standard Buffer (condition 1)4DNES1QS8EMG4DNESURW4RWH2Per replicate Normalized counts
\n
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The middle track uses an antibody that detects a poised version of RNA Pol II subunit as a marker of actively transcribed regions. While the bottom 2 tracks are for Lamins markers of the nuclear lamina.\n\nThe display below highlights the finding that actively transcribing regions and nuclear speckles tend to have similar TSA-seq profiles that are generally inverse to profiles using markers for the more inactive lamina regions. 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