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Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Mich, John K. and Graybuck, Lucas T. and Hess, Erik E. and Mahoney, Joseph T. and Kojimo, Yoshiko and Ding, Yi and Somasundaram, Saroja and Miller, Jeremy A. and Weed, Natalie and Omstead, Victoria and Bishaw, Yemeserach and Shapovalova, Nadiya V. and Martinez, Refugio A. and Fong, Olivia and Yao, Shenqin and Mortrud, Marty and Chong, Peter and Loftus, Luke and Bertagnolli, Darren and Goldy, Jeff and Casper, Tamara and Dee, Nick and Opitz-Araya, Ximena and Cetin, Ali and Smith, Kimberly A. and Gwinn, Ryder P. and Cobbs, Charles and Ko, Andrew L. and Ojemann, Jeffrey G. and Keene, C. Dirk and Silbergeld, Daniel L. and Sunkin, Susan M. and Gradinaru, Viviana and Horwitz, Gregory D. and Zeng, Hongkui and Tasic, Bosiljka and Lein, Ed S. and Ting, Jonathan T. and Levi, Boaz P. (2020) Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20200421-093728277

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Abstract

Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/555318DOIDiscussion Paper
ORCID:
AuthorORCID
Gradinaru, Viviana0000-0001-5868-348X
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. Posted April 21, 2020. We thank Allison Beller, Nathan Hansen, Caryl Tongco, Jae-Guen Yoon, and Gina DeNoble for assistance with obtaining patient consent and human neurosurgical tissue research specimens. We thank Rebecca D. Hodge and Trygve E. Bakken and Zizhen Yao for assistance with sc/snRNA-seq data. We thank Lisa McConnell for assisting with NHP virus injection surgery and also NHP animal care. We thank Allen Institute Tissue Procurement and Facilities teams for institutional support during tissue collections. Funding: This work is supported by NIH BRAIN Initiative award #1RF1MH114126-01 from the National Institute of Mental Health to ESL, JTT, and BPL, and National Institute on Drug Abuse award #1R01DA036909-01 to BT, the Nancy and Buster Alvord Endowment to CDK, National Eye Institute award # 1R01EY030441-01 to GDH. Also, this project was supported in part by NIH grants P51OD010425 from the Office of Research Infrastructure Programs (ORIP) and UL1TR000423 from the National Center for Advancing Translational Sciences (NCATS). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NIH, ORIP, NCATS, the Institute of Translational Health Sciences or the University of Washington National Primate Research Center.” In addition, we wish to thank the Allen Institute for Brain Science founder, Paul G. Allen, for his vision, encouragement and support. Author contributions: JTT, NS, EEH, TC, ND, and JKM performed tissue processing and flow cytometry. EEH, BPL, and JKM performed ATAC-seq with assistance from DB and KAS. JKM analyzed ATAC-seq data using techniques developed by LTG, with assistance from SS, JAM, LTG, JG, and YD. BPL, JTT, and JKM curated candidate enhancers for testing. EEH, JKM, JTT, RAM, and XOA performed AAV vector design and molecular biology. JKM and JTT tested AAV vectors with assistance from PC and XOA. RPG, CC, JGO, ALK, CDK, and DLS procured human surgical tissue for research. JTT and JKM performed human ex vivo brain slice culture and viral labeling experiments with assistance from PC. JTM, LL and YD performed mFISHHCR. Single cell RNA-seq was conducted by JKM, DB, KAS, and analysis by OF, JG, and BPL. MM, SY, AC, EEH, and XOA performed viral packaging. JTT carried out non-human primate ex vivo slice culture experiments with assistance from GDH, NW, and XOA. GDH and YK performed NHP in vivo virus injection surgery with assistance from JTT. JKM and JTT processed NHP brain tissue from in vivo virus testing with assistance from VO and YB. BPL, JTT, JKM, and EL conceived of the study design. JKM wrote the manuscript and prepared figures. LTG and BT provided mouse ATAC-seq data. VG provided PHP.eB capsid plasmid DNA. SMS provided program and budgetary management. HZ and ESL provided program leadership. Competing interests: JKM, LTG, EEH, HZ, BT, EL, JTT, and BPL are inventors on several U.S. provisional patent applications related to this work. All authors declare no other competing interests. Data and materials availability: scATAC-seq and scRNA-seq data will be deposited to GEO or dbGAP. Software code used for data analysis and visualization is available from GitHub at https://github.com/AllenInstitute/graybuck2019analysis/.
Funders:
Funding AgencyGrant Number
NIH1RF1MH114126-01
NIH1R01DA036909-01
Nancy and Buster Alvord EndowmentUNSPECIFIED
NIH1R01EY030441-01
NIHP51OD010425
NIHUL1TR000423
Record Number:CaltechAUTHORS:20200421-093728277
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200421-093728277
Official Citation:Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. John K. Mich, Lucas T. Graybuck, Erik E. Hess, Joseph T. Mahoney, Yoshiko Kojimo, Yi Ding, Saroja Somasundaram, Jeremy A. Miller, Natalie Weed, Victoria Omstead, Yemeserach Bishaw, Nadiya V. Shapovalova, Refugio A. Martinez, Olivia Fong, Shenqin Yao, Marty Mortrud, Peter Chong, Luke Loftus, Darren Bertagnolli, Jeff Goldy, Tamara Casper, Nick Dee, Ximena Opitz-Araya, Ali Cetin, Kimberly A. Smith, Ryder P. Gwinn, Charles Cobbs, Andrew L. Ko, Jeffrey G. Ojemann, C. Dirk Keene, Daniel L. Silbergeld, Susan M. Sunkin, Viviana Gradinaru, Gregory D. Horwitz, Hongkui Zeng, Bosiljka Tasic, Ed S. Lein, Jonathan T. Ting, Boaz P. Levi. bioRxiv 555318; doi: https://doi.org/10.1101/555318
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102694
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Apr 2020 17:04
Last Modified:21 Apr 2020 17:04

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