{"lab": {"uuid": "3452dfa5-ee8b-4dbc-8068-00cadde268b9", "@type": ["Lab", "Item"], "title": "Sheng Zhong, UCSD", "status": "current", "correspondence": [{"contact_email": "c3pob25nQHVjc2QuZWR1", "@id": "/users/5549e16d-fa63-477c-a1ed-6189cecc1304/", "display_title": "Sheng Zhong"}], "@id": "/labs/sheng-zhong-lab/", "display_title": "Sheng Zhong, UCSD", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.3452dfa5-ee8b-4dbc-8068-00cadde268b9"]}}, "award": {"name": "2U01CA200147-06", "@type": ["Award", "Item"], "description": "OH: The second phase of the 4DN program requires an efficient organizational center to synergize the activities of all the funded teams and integrate the research products. 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Our major deliverables are: (1) an organizational structure composed of a steering committee and problem-solving working groups with a clear report chain to enable effective communications and decision-making processes, establish co-organizers and schedule, clarify action items, and ensure execution of the action items; (2) 4DN Web Portal (https://www.4dnucleome.org/), which is the always up-to-date community-wide resource and point of access for all data, protocols, reagents, resources, and methods; (3) 4DN internal wiki, the central organized resource for all teams, centers, and working groups to document internal progresses. (4) 4DN annual meetings, including the \"\"kick-off\"\" meeting in winter 2020 and the subsequent annual meetings and 4DN-ASCB (American Society of Cell Biology) satellite meetings; (5) 4DN outreach workshops at Keystone symposia and American Society of Human Genetics (ASHG) meetings, and on 4DN YouTube channel.", "@id": "/awards/2U01CA200147-06/", "center_title": "OH - Zhong", "status": "current", "project": "4DN", "uuid": "1a4da807-8221-47d2-96ab-7a31485974c3", "display_title": "THE SECOND PHASE OF NIH COMMON FUND 4D NUCLEOME NETWORK ORGANIZATIONAL HUB", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "status": "released", "aliases": ["sheng-zhong-lab:iMARGI-on-hff"], "accession": "4DNES9Y1GHK4", "condition": "No treatment control", "description": "iMARGI on hTert-immortalized human foreskin fibroblasts", "date_created": "2021-07-01T17:48:42.072714+00:00", "submitted_by": {"error": "no view permissions"}, "dataset_label": "iMARGI on HFFc6 cells", "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2021-12-17T00:46:31.184623+00:00"}, "public_release": "2021-07-27", "replicate_exps": [{"bio_rep_no": 1, "tec_rep_no": 1, "replicate_exp": {"display_title": "MARGI on HFFc6 (Tier 1) - 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\n\n

\n Image Source: Weixin Wu, Xingzhao Wen, Sheng Zhong, private communication \n
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\n\n

\n Image Source: Weixin Wu, Xingzhao Wen, Sheng Zhong, private communication \n
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MARGI

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MARGI is a method to map native RNA-DNA interactions from unperturbed cells at a global scale. It is a method analogous to the measurement of DNA-DNA interactions by Hi-C experiments. MARGI was first published in 2017, and since then, the protocol has been rapidly improved. MARGI 2.0 is the latest version that introduced RNA-DNA proximity ligation in situ inside intact nuclei instead of in solution in order to reduce the rate of random ligation. In addition, this version also reduces the necessary amount of cell input 100-fold and shortens experimental processing time.

\n

The protocol involves crosslinking the cells with formaldehyde to form links between physically adjacent RNA-DNA molecules. The intact nuclei are collected from fixed cells and permeabilized with SDS. A restriction enzyme (AluI) is used to digest chromatin into DNA fragments and RNase I is used to fragment RNA. A specially designed biotinylated linker is introduced to ligate to 3\u00b4-end of RNA first and then this RNA-ligated linker is ligated to DNA. These ligation steps are controlled by the configuration of the linker and by sequential applications of different end modifications and ligation enzymes. Nuclei are lysed, and successfully ligated products, in the form of RNA-linker-DNA, are selected with streptavidin beads, followed by converting the RNA part of the chimera into cDNA on beads. cDNA-linker-DNA is circularized and then cut in the middle, producing DNAs with the configuration left.half.Linker-RNA-DNA-right.half.Linker, which are subjected to library amplification and paired-end sequencing.

\n
\n\n

\n Image Source: Weixin Wu, Xingzhao Wen, Sheng Zhong, private communication \n
\n\n
\n
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This experiment set supersedes 4DNESNGESP28 and 4DNESJC437VS because the replicate structure of the old set was determined to be incorrect as submitted. Experiments submitted as technical replicates were in fact biological replicates. Two of the technical replicates that corresponding to miseq QC runs have been removed and additional new sequencing replicates (i.e. new fastq files) have been added to each biological replicate experiment of the superseding replicate set. Due to the change in replicate structure new accessions have been assigned for experiments and biosamples, however, accessions for unchanged files are shared between the new and replaced replicate sets..

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We find that the relative abundance of trans-acting caRNA reflects the functional nuclear compartmentalization of the 3D genome, and the caRNAs sequence features are predictive of its spatial localization. We observe extensive caRNA-genome interactions that span several hundred kilobases to several megabases. These caRNA domains correlate with chromatin loops and enhancer-promoter interactions. We report a tradeoff of caRNAs promotive and suppressive roles to chromatin interactions. As a result, caRNA promotes chromatin interactions outside the anchors of chromatin loops, whereas caRNA exhibits an overall suppressive impact to chromatin interactions between loop anchors. Furthermore, caRNA suppresses chromatin loops number and strengths genome-wide, and this suppression depends on electric charge-mediated RNA interactions. These results reveal caRNAs multifaced role in regulating 3D chromatin organization and highlight caRNAs ability to modulate chromatin looping.", "@id": "/publications/d313c2a4-aab7-417a-b9b4-bcf5ec5cdbac/", "@type": ["Publication", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "pubs_using": [], "publications_of_set": [{"title": "Three-dimensional organization of chromatin associated RNAs and their role in chromatin architecture in human cells", "@id": "/publications/d313c2a4-aab7-417a-b9b4-bcf5ec5cdbac/", "uuid": "d313c2a4-aab7-417a-b9b4-bcf5ec5cdbac", "abstract": "Chromatin-associated RNA (caRNA) is a vital component of the interphase nucleus; yet its distribution and role in the three-dimensional (3D) genome organization and function remain poorly understood. Here, we map caRNAs spatial distribution on the 3D genome in human embryonic stem cells, fibroblasts, and myelogenous leukemia cells. We find that the relative abundance of trans-acting caRNA reflects the functional nuclear compartmentalization of the 3D genome, and the caRNAs sequence features are predictive of its spatial localization. We observe extensive caRNA-genome interactions that span several hundred kilobases to several megabases. These caRNA domains correlate with chromatin loops and enhancer-promoter interactions. We report a tradeoff of caRNAs promotive and suppressive roles to chromatin interactions. As a result, caRNA promotes chromatin interactions outside the anchors of chromatin loops, whereas caRNA exhibits an overall suppressive impact to chromatin interactions between loop anchors. Furthermore, caRNA suppresses chromatin loops number and strengths genome-wide, and this suppression depends on electric charge-mediated RNA interactions. 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