{"lab": {"title": "Karla Neugebauer, YALE", "@type": ["Lab", "Item"], "status": "current", "correspondence": [{"contact_email": "a2FybGEubmV1Z2ViYXVlckB5YWxlLmVkdQ==", "@id": "/users/3274e33f-c3d2-4774-91ce-e8877dba6aa6/", "display_title": "Karla Neugebauer"}], "display_title": "Karla Neugebauer, YALE", "@id": "/labs/karla-neugebauer-lab/", "uuid": "1eea19ac-724d-41f3-a779-623df2db5f22", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.1eea19ac-724d-41f3-a779-623df2db5f22"]}}, "award": {"center_title": "TCPA - Neugebauer", "display_title": "THE CELL NUCLEUS UNDER STRESS", "description": "TCPA: Environmental stress alters the organization and function of the cell nucleus. Stress induces dramatic shifts in the transcriptional activity and nuclear positioning of genes, suggesting extensive remodeling of the genome upon heat and osmotic shock, UV damage, viral infection, hypoxia, etc. Moreover, membraneless nuclear bodies, such as nucleoli and Cajal bodies (CBs) \u2013 nucleated by low complexity (LC) proteins at specific chromosomal loci \u2013 are disrupted. These loci cluster in nuclear bodies in unstressed cells, raising the possibility that stress \ninduces widespread changes in chromosome topology. Despite this evidence, a systematic investigation of how stress changes the 4D nuclear landscape has not been undertaken. \nOur labs are expert in the investigation of nuclear bodies and of long non-coding RNAs (lncRNAs). The Neugebauer lab has shown that nuclear bodies form at active sites of transcription and that LC proteins, such as coilin in CBs, can be ChIPped at gene loci and crosslinked to the corresponding RNAs in vivo. Thus, our methods use DNA and RNA sequencing to pinpoint genomic location(s) of nuclear bodies at molecular resolution. The \nSteitz lab has recently discovered thousands of novel lncRNAs produced by transcriptional read-through of protein-coding genes. These DoGs (Downstream of Gene transcripts) \u2013induced by osmotic stress via the IP3 receptor pathway \u2013 are up to 45kb in length and >2000 in number in human cells. Also induced by heat and oxidative stress, DoGs thereby define transcriptionally active \u201cintergenic\u201d regions that likely contribute to and/or result from changes in the 4D organization of the genome under stress. Using heat, osmotic and oxidative stress as models, we propose to identify changes in 1) chromosome topology through Hi-C, 4C, and ChIA-PET, 2) regions of genome activity through Transient Transcriptome (TT)-Seq, ChIP-Seq and development of TT-CLIP to monitor LC protein interactions with DNA and RNA at high spatial and temporal resolution, and 3) imaging of nuclear bodies and clustered genomic loci using super-resolution fluorescence microscopy. The functional significance of nuclear bodies and DoGs will be tested during stress and recovery. A unifying theme is that LC proteins and lncRNAs are unconventional molecular species with emerging roles in genome organization. Our preliminary data show the induction of DoGs and disruption of CBs over time in human and mouse cells. Intriguingly, the CB component SMN \u2013 an LC protein implicated in transcription termination \u2013 remains in nuclear bodies upon osmotic stress only, suggesting differential functions in stress responses. All experiments will be conducted in human tissue culture cells to maximize compatibility with other 4DN projects. Our project contributes novelty and innovation to the 4DN Initiative through: the use of RNA and DNA sequencing data to identify genomic regions of interest, our commitment to uncovering comprehensive changes in genome organization due to stress, our interest in a novel class of lncRNAs (DoGs), and our ability to bridge sequencing and imaging approaches.", "uuid": "e9b7d287-40b3-44b6-9a10-be28cf2347f0", "status": "current", "name": "TCPA-2017-06", "@id": "/awards/TCPA-2017-06/", "project": "4DN", "@type": ["Award", "Item"], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "files": [{"display_title": "4DNFIWODP7EQ.tiff", "file_type_detailed": "time-series (tiff)", "href": "/files-microscopy/4DNFIWODP7EQ/@@download/4DNFIWODP7EQ.tiff", "status": "released", "uuid": "0a7e7eb9-9ecf-407c-81eb-5efb47589f89", "@type": ["FileMicroscopy", "File", "Item"], "@id": "/files-microscopy/4DNFIWODP7EQ/", "file_classification": "raw 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