CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function.
CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Ya
Y
Guo
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Quan
Q
Xu
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, 701 West 168(th) Street, New York, NY 10032, USA.
Daniele
D
Canzio
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Jia
J
Shou
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Jinhuan
J
Li
Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA.
David U
DU
Gorkin
Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Inkyung
I
Jung
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Haiyang
H
Wu
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Yanan
Y
Zhai
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Yuanxiao
Y
Tang
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Yichao
Y
Lu
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Yonghu
Y
Wu
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Zhilian
Z
Jia
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China.
Wei
W
Li
Department of Molecular and Cell Biology, Center for Systems Biology, University of Texas at Dallas, Richardson, TX 75080, USA; MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, China.
Michael Q
MQ
Zhang
Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Bing
B
Ren
Cold Spring Harbor Laboratory, 1 Bungtown Rd, NY 11724, USA.
Adrian R
AR
Krainer
Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, 701 West 168(th) Street, New York, NY 10032, USA. Electronic address: tm2472@cumc.columbia.edu.
Tom
T
Maniatis
Center for Comparative Biomedicine, MOE Key Laboratory of Systems Biomedicine, Institute of Systems Biomedicine, Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University (SJTU), Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, SJTU, Shanghai 200240, China; Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (MOE), Bio-X Center, School of Life Sciences and Biotechnology, SJTU, Shanghai 200240, China. Electronic address: qwu123@gmail.com.
Qiang
Q
Wu
D000818:Animals; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D015820:Cadherins / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D002875:Chromosomes / Q000737:chemistry / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005821:Genetic Techniques; D015894:Genome, Human; D006801:Humans; D020014:K562 Cells; D051379:Mice; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D055544:beta-Globins / Q000235:genetics; D000097722:Cohesins
D000076246:CCCTC-Binding Factor; D015820:Cadherins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004247:DNA; D012097:Repressor Proteins; D055544:beta-Globins; D000097722:Cohesins; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse