Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements
To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure t...
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| Veröffentlicht in: | Science (American Association for the Advancement of Science) 2018-03, Vol.359 (6379), p.1050-1055 |
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| creator | Gu, Bo Swigut, Tomek Spencley, Andrew Bauer, Matthew R Chung, Mingyu Meyer, Tobias Wysocka, Joanna |
| description | To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription. |
| doi_str_mv | 10.1126/science.aao3136 |
| format | Article |
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We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.</description><identifier>ISSN: 0036-8075</identifier><identifier>EISSN: 1095-9203</identifier><identifier>DOI: 10.1126/science.aao3136</identifier><identifier>PMID: 29371426</identifier><language>eng</language><publisher>United States: The American Association for the Advancement of Science</publisher><subject>Animals ; Bacterial Proteins ; Cargo ; Cell differentiation ; Cell Line ; Cell Nucleus - genetics ; Cells (biology) ; CRISPR-Associated Protein 9 ; Deoxyribonucleic acid ; Differentiation (biology) ; DNA ; DNA-directed RNA polymerase ; Embryo cells ; Endonucleases ; Enhancers ; Gene regulation ; gRNA ; Individualized Instruction ; Loci ; Mice ; Mobility ; Multiplexing ; Nuclei ; Nuclei (cytology) ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; Promoters ; Regulatory sequences ; Regulatory Sequences, Nucleic Acid ; Ribonucleic acid ; RNA ; RNA polymerase II ; RNA Polymerase II - metabolism ; RNA, Guide, CRISPR-Cas Systems ; Single Molecule Imaging - methods ; Single-Cell Analysis - methods ; Stem cells ; Transcription activation ; Transcription, Genetic ; Transcriptional Activation</subject><ispartof>Science (American Association for the Advancement of Science), 2018-03, Vol.359 (6379), p.1050-1055</ispartof><rights>Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.</rights><rights>Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c524t-cc5efd4bf45ebb52bd38099fe9d3f0983ad524385162c63125d5afaa64ed590f3</citedby><cites>FETCH-LOGICAL-c524t-cc5efd4bf45ebb52bd38099fe9d3f0983ad524385162c63125d5afaa64ed590f3</cites><orcidid>0000-0002-6909-6544 ; 0000-0002-6711-4139 ; 0000-0002-7746-6015 ; 0000-0003-1718-6203</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,2884,2885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29371426$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gu, Bo</creatorcontrib><creatorcontrib>Swigut, Tomek</creatorcontrib><creatorcontrib>Spencley, Andrew</creatorcontrib><creatorcontrib>Bauer, Matthew R</creatorcontrib><creatorcontrib>Chung, Mingyu</creatorcontrib><creatorcontrib>Meyer, Tobias</creatorcontrib><creatorcontrib>Wysocka, Joanna</creatorcontrib><title>Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements</title><title>Science (American Association for the Advancement of Science)</title><addtitle>Science</addtitle><description>To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). 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Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.</description><subject>Animals</subject><subject>Bacterial Proteins</subject><subject>Cargo</subject><subject>Cell differentiation</subject><subject>Cell Line</subject><subject>Cell Nucleus - genetics</subject><subject>Cells (biology)</subject><subject>CRISPR-Associated Protein 9</subject><subject>Deoxyribonucleic acid</subject><subject>Differentiation (biology)</subject><subject>DNA</subject><subject>DNA-directed RNA polymerase</subject><subject>Embryo cells</subject><subject>Endonucleases</subject><subject>Enhancers</subject><subject>Gene regulation</subject><subject>gRNA</subject><subject>Individualized Instruction</subject><subject>Loci</subject><subject>Mice</subject><subject>Mobility</subject><subject>Multiplexing</subject><subject>Nuclei</subject><subject>Nuclei (cytology)</subject><subject>Oligonucleotide Array Sequence Analysis</subject><subject>Oligonucleotides</subject><subject>Promoters</subject><subject>Regulatory sequences</subject><subject>Regulatory Sequences, Nucleic Acid</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA polymerase II</subject><subject>RNA Polymerase II - metabolism</subject><subject>RNA, Guide, CRISPR-Cas Systems</subject><subject>Single Molecule Imaging - methods</subject><subject>Single-Cell Analysis - methods</subject><subject>Stem cells</subject><subject>Transcription activation</subject><subject>Transcription, Genetic</subject><subject>Transcriptional Activation</subject><issn>0036-8075</issn><issn>1095-9203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc1LxDAQxYMoun6cvUnBi5dq0jTd5iKI-AWCF_UapulkjaTJmrTC_vdGXEU9zWF-82bePEIOGT1lrGrOkrboNZ4CBM54s0FmjEpRyoryTTKjlDdlS-dih-ym9Epp7km-TXYqyeesrpoZeX6M4JOOdjna4EsdpqXDvtAv4BeYCusLP2mHEIshdNbZcVUEUwwwDOAs-ELbVEZcTA7GEFcFOhzQj2mfbBlwCQ_WdY88XV89Xt6W9w83d5cX96UWVT2WWgs0fd2ZWmDXiarreUulNCh7bqhsOfSZ461gTaUbzirRCzAATY29kNTwPXL-pbucugF7nXdHcGoZ7QBxpQJY9bfj7YtahHfV5HHB2ixwshaI4W3CNKrBJo3OgccwJcVk_iUTc15n9Pgf-hqm6LM9lRHaZsk5zdTZF6VjSCmi-TmGUfWZmVpnptaZ5Ymj3x5--O-Q-Ae7Mpd2</recordid><startdate>20180302</startdate><enddate>20180302</enddate><creator>Gu, Bo</creator><creator>Swigut, Tomek</creator><creator>Spencley, Andrew</creator><creator>Bauer, Matthew R</creator><creator>Chung, Mingyu</creator><creator>Meyer, Tobias</creator><creator>Wysocka, Joanna</creator><general>The American Association for the Advancement of Science</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QQ</scope><scope>7QR</scope><scope>7SC</scope><scope>7SE</scope><scope>7SN</scope><scope>7SP</scope><scope>7SR</scope><scope>7SS</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7TK</scope><scope>7TM</scope><scope>7U5</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>K9.</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-6909-6544</orcidid><orcidid>https://orcid.org/0000-0002-6711-4139</orcidid><orcidid>https://orcid.org/0000-0002-7746-6015</orcidid><orcidid>https://orcid.org/0000-0003-1718-6203</orcidid></search><sort><creationdate>20180302</creationdate><title>Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements</title><author>Gu, Bo ; Swigut, Tomek ; Spencley, Andrew ; Bauer, Matthew R ; Chung, Mingyu ; Meyer, Tobias ; Wysocka, Joanna</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c524t-cc5efd4bf45ebb52bd38099fe9d3f0983ad524385162c63125d5afaa64ed590f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Animals</topic><topic>Bacterial Proteins</topic><topic>Cargo</topic><topic>Cell differentiation</topic><topic>Cell Line</topic><topic>Cell Nucleus - genetics</topic><topic>Cells (biology)</topic><topic>CRISPR-Associated Protein 9</topic><topic>Deoxyribonucleic acid</topic><topic>Differentiation (biology)</topic><topic>DNA</topic><topic>DNA-directed RNA polymerase</topic><topic>Embryo cells</topic><topic>Endonucleases</topic><topic>Enhancers</topic><topic>Gene regulation</topic><topic>gRNA</topic><topic>Individualized Instruction</topic><topic>Loci</topic><topic>Mice</topic><topic>Mobility</topic><topic>Multiplexing</topic><topic>Nuclei</topic><topic>Nuclei (cytology)</topic><topic>Oligonucleotide Array Sequence Analysis</topic><topic>Oligonucleotides</topic><topic>Promoters</topic><topic>Regulatory sequences</topic><topic>Regulatory Sequences, Nucleic Acid</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA polymerase II</topic><topic>RNA Polymerase II - metabolism</topic><topic>RNA, Guide, CRISPR-Cas Systems</topic><topic>Single Molecule Imaging - methods</topic><topic>Single-Cell Analysis - methods</topic><topic>Stem cells</topic><topic>Transcription activation</topic><topic>Transcription, Genetic</topic><topic>Transcriptional Activation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gu, Bo</creatorcontrib><creatorcontrib>Swigut, Tomek</creatorcontrib><creatorcontrib>Spencley, Andrew</creatorcontrib><creatorcontrib>Bauer, Matthew R</creatorcontrib><creatorcontrib>Chung, Mingyu</creatorcontrib><creatorcontrib>Meyer, Tobias</creatorcontrib><creatorcontrib>Wysocka, Joanna</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Ecology Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Science (American Association for the Advancement of Science)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gu, Bo</au><au>Swigut, Tomek</au><au>Spencley, Andrew</au><au>Bauer, Matthew R</au><au>Chung, Mingyu</au><au>Meyer, Tobias</au><au>Wysocka, Joanna</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements</atitle><jtitle>Science (American Association for the Advancement of Science)</jtitle><addtitle>Science</addtitle><date>2018-03-02</date><risdate>2018</risdate><volume>359</volume><issue>6379</issue><spage>1050</spage><epage>1055</epage><pages>1050-1055</pages><issn>0036-8075</issn><eissn>1095-9203</eissn><abstract>To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. 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| subjects | Animals Bacterial Proteins Cargo Cell differentiation Cell Line Cell Nucleus - genetics Cells (biology) CRISPR-Associated Protein 9 Deoxyribonucleic acid Differentiation (biology) DNA DNA-directed RNA polymerase Embryo cells Endonucleases Enhancers Gene regulation gRNA Individualized Instruction Loci Mice Mobility Multiplexing Nuclei Nuclei (cytology) Oligonucleotide Array Sequence Analysis Oligonucleotides Promoters Regulatory sequences Regulatory Sequences, Nucleic Acid Ribonucleic acid RNA RNA polymerase II RNA Polymerase II - metabolism RNA, Guide, CRISPR-Cas Systems Single Molecule Imaging - methods Single-Cell Analysis - methods Stem cells Transcription activation Transcription, Genetic Transcriptional Activation |
| title | Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements |
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