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Parallel functional testing identifies enhancers active in early postnatal mouse brain

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Cite this article as: eLife 2021;10:e69479 doi: 10.7554/eLife.69479

Abstract

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

Data availability

All supplementary information, including links to raw and processed data, can be found at the Nord Lab Resources page (https://nordlab.faculty.ucdavis.edu/resources/). Software can be found at the Nord Lab Git Repository (https://github.com/NordNeurogenomicsLab/) and https://github.com/NordNeurogenomicsLab/Publications/tree/master/Lambert_eLIFE_2021. Sequencing data have been deposited in GEO under accession code GSE172058.

The following data sets were generated
The following previously published data sets were used
    1. Roadmap Epigenomics Consortium
    (2013) Roadmap Consolidated Peak Dataset
    GEO GSM530651, GSM595913, GSM595920, GSM595922, GSM595923, GSM595926, GSM595928, GSM806934, GSM806939, GSM621457, GSM706999, GSM806935, GSM621427, GSM707000, GSM806936, GSM621393, GSM707001, GSM806937, GSM621410, GSM707002, GSM806938.

Article and author information

Author details

  1. Jason T Lambert

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Linda Su-Feher

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Karol Cichewicz

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5926-3663
  4. Tracy L Warren

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5125-0868
  5. Iva Zdilar

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0080-3132
  6. Yurong Wang

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Kenneth J Lim

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Jessica L Haigh

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9518-4003
  9. Sarah J Morse

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Cesar P Canales

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2505-8367
  11. Tyler W Stradleigh

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Erika Castillo Palacios

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Viktoria Haghani

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3700-4027
  14. Spencer D Moss

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Hannah Parolini

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Diana Quintero

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Diwash Shrestha

    University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Daniel Vogt

    Department of Pediatrics & Human Development, Michigan State University, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
  19. Leah C Byrne

    University of Pittsburgh, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  20. Alex S Nord

    University of California, Davis, Davis, United States
    For correspondence
    asnord@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4259-7514

Funding

National Institutes of Health (R35GM119831)

  • Jason T Lambert

National Institutes of Health (T32-GM008799)

  • Linda Su-Feher

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All procedures were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of California Animal Care and Use Committee (AUP #R200-0913BC). Surgery was performed under anesthesia, and all efforts were made to minimize suffering.

Reviewing Editor

  1. Genevieve Konopka, University of Texas Southwestern Medical Center, United States

Publication history

  1. Received: April 19, 2021
  2. Accepted: October 2, 2021
  3. Accepted Manuscript published: October 4, 2021 (version 1)

Copyright

© 2021, Lambert et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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    Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution – as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.