Abstract

Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHD). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.

Data availability

Sequencing data have been deposited in GEO under accession code: GSE190475.

The following data sets were generated

Article and author information

Author details

  1. Daniel A Armendariz

    Cecil H and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Sean C Goetsch

    Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Anjana Sundarrajan

    Cecil H and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Sushama Sivakumar

    Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7877-4821
  5. Yihan Wang

    Cecil H and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Shiqi Xie

    Cecil H and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Nikhil V Munshi

    Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Nikhil.Munshi@UTSouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
  8. Gary C Hon

    Cecil H and Ida Green Center for Reproductive Biology Sciences, The University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Gary.Hon@UTSouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1615-0391

Funding

NIH (DP2GM128203)

  • Gary C Hon

Department of Defense (PR172060)

  • Nikhil V Munshi

NIH (UM1HG011996)

  • Nikhil V Munshi
  • Gary C Hon

NIH (1R35GM145235)

  • Gary C Hon

CPRIT (RP190451)

  • Gary C Hon

NIH (HL136604)

  • Nikhil V Munshi

NIH (HL151650)

  • Nikhil V Munshi

Burroughs Wellcome Fund (1019804)

  • Gary C Hon

Burroughs Wellcome Fund (1009838)

  • Nikhil V Munshi

Welch Foundation (I-2103-2022033)

  • Gary C Hon

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

Reviewing Editor

  1. Benoit Bruneau, University of California, San Francisco, United States

Version history

  1. Preprint posted: April 10, 2022 (view preprint)
  2. Received: January 15, 2023
  3. Accepted: March 10, 2023
  4. Accepted Manuscript published: April 25, 2023 (version 1)
  5. Version of Record published: May 3, 2023 (version 2)

Copyright

© 2023, Armendariz 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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  1. Daniel A Armendariz
  2. Sean C Goetsch
  3. Anjana Sundarrajan
  4. Sushama Sivakumar
  5. Yihan Wang
  6. Shiqi Xie
  7. Nikhil V Munshi
  8. Gary C Hon
(2023)
CHD-associated enhancers shape human cardiomyocyte lineage commitment
eLife 12:e86206.
https://doi.org/10.7554/eLife.86206

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