{"lab": {"uuid": "c17e88b5-912a-496a-acc7-28dd71215a7d", "@type": ["Lab", "Item"], "@id": "/labs/mitchell-guttman-lab/", "title": "Mitchell Guttman, CALTECH", "status": "current", "display_title": "Mitchell Guttman, CALTECH", "correspondence": [{"contact_email": "bWd1dHRtYW5AY2FsdGVjaC5lZHU=", "@id": "/users/ac3920c8-caa6-444b-be8a-48b52a1dcb3e/", "display_title": "Mitchell Guttman"}], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.c17e88b5-912a-496a-acc7-28dd71215a7d"]}}, "award": {"status": "current", "@type": ["Award", "Item"], "@id": "/awards/1U01DA040612-01/", "uuid": "4de1cf81-1ea0-4a19-bc7a-dd3343b34438", "center_title": "NBC - Guttman", "display_title": "DECIPHERING THE FUNCTION AND MECHANISMS OF LNCRNA-MEDIATED ORGANIZATION OF NUCLEAR COMPARTMENTS", "description": "NBC: The nucleus of each cell is a complex arrangement of DNA, RNA, and protein that is dynamically organized into various nuclear bodies and compartments that are often arranged around shared functional and regulatory roles. Yet, while many of these nuclear compartments were first identified several decades ago a major challenge with characterizing these compartments is that there are currently no biochemical methods for isolating individual nuclear compartments. Importantly, many nuclear bodies are marked and maintained by nuclear retained long non-coding RNAs (lncRNAs). Here we aim to develop several novel technologies to purify the molecular constituents of nuclear domains and their necessity and sufficiency in forming these compartments. Together these technologies will allow us to systematically address the following questions: Aim 1: What are the optimal biochemical conditions in which to specifically and accurately purify specific nuclear bodies and compartments in order to identify their DNA, RNA and protein components? A critical aspect of biochemical purifications is the fine balance between cross-linking conditions that identify direct molecular interactions while not over-crosslinking that could result in indirect interactions. Usin known nuclear compartments such as the nucleolus, nuclear speckles, and paraspeckles we will optimize RAP for isolating DNA-RNA, RNA-RNA, RNA-protein interactions. We aim to identify universally applicable purification conditions to identify molecular interactions within nuclear and other subcellular structures. Aim 2: What are the DNA, RNA and protein factors involved in nuclear compartments? Although most known nuclear bodies are characterized by lncRNAs the other RNA, DNA and protein factors remain less well defined. Here we will develop technologies to catalog the molecular factors that comprise various nuclear bodies. We will further validate these components through colocalization of these components within a nuclear domain using visualization approaches (e.g. RNA-DNA Co-FISH). Overall we aim to apply new technologies to systematic and comprehensive catalog of RNA, DNA and Proteins in nuclear bodies. Aim 3: Are lncRNAs and proteins necessary and/or sufficient for nuclear compartmentalization? Here we will develop a novel technology platform, termed CRISPR-Display, which allows long RNA cargos to be appended and delivered by CRISPR-Cas9 systems to a desired site in the genome. We will develop this technology to test if lncRNA molecules are sufficient to drive nuclear organization. We will also use CRISPR-Display to multiplex several RNA aptamers that can be used to recruit proteins (with reciprocal protein epitopes that bind RNA aptamers) and test if they are sufficient to form nuclear compartments. In parallel we will perform loss-of- function approaches to identify protein and or RNA components are required for establishing nuclear domains. 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DNA FISH
\nDNA FISH is a method to detect the position of specific DNA regions in the cell through fluorescent labeling. It can be used to measure the 3D distances between multiple genomic loci. Additionally, it is often used to corroborate the findings of chromosome capture techniques (3C).
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We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.", "@id": "/publications/4ab311b1-a442-46b4-ae4f-16c57521f52e/", "status": "current", "authors": ["Quinodoz SA", "Ollikainen N", "Tabak B", "Palla A", "Schmidt JM", "Detmar E", "Lai MM", "Shishkin AA", "Bhat P", "Takei Y", "Trinh V", "Aznauryan E", "Russell P", "Cheng C", "Jovanovic M", "Chow A", "Cai L", "McDonel P", "Garber M", "Guttman M"], "title": "Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.", "ID": "doi:10.1016/j.cell.2018.05.024", "short_attribution": "Quinodoz SA et al. (2018)", "url": "https://www.ncbi.nlm.nih.gov/pubmed/29887377", "journal": "Cell", "@type": ["Publication", "Item"], "date_published": "2018-06-07", "uuid": "4ab311b1-a442-46b4-ae4f-16c57521f52e", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "pubs_using": [], "publications_of_set": [{"@type": ["Publication", "Item"], "@id": "/publications/4ab311b1-a442-46b4-ae4f-16c57521f52e/", "date_published": "2018-06-07", "ID": "doi:10.1016/j.cell.2018.05.024", "authors": ["Quinodoz SA", "Ollikainen N", "Tabak B", "Palla A", "Schmidt JM", "Detmar E", "Lai MM", "Shishkin AA", "Bhat P", "Takei Y", "Trinh V", "Aznauryan E", "Russell P", "Cheng C", "Jovanovic M", "Chow A", "Cai L", "McDonel P", "Garber M", "Guttman M"], "abstract": "Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.", "title": "Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.", "status": "current", "display_title": "Quinodoz SA et al. 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