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CHD-associated enhancers shape human cardiomyocyte lineage commitment

Daniel A. Armendariz, Sean C. Goetsch, Anjana Sundarrajan, Sushama Sivakumar, Yihan Wang, Shiqi Xie, Nikhil Munshi, View ORCID ProfileGary C. Hon
doi: https://doi.org/10.1101/2022.04.07.487515
Daniel A. Armendariz
1Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Sean C. Goetsch
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Anjana Sundarrajan
1Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Sushama Sivakumar
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Yihan Wang
1Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Shiqi Xie
1Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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Nikhil Munshi
2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
3Division of Cardiology, Department of Molecular Biology, McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
4Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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  • For correspondence: Nikhil.Munshi@utsouthwestern.edu Gary.Hon@utsouthwestern.edu
Gary C. Hon
1Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
4Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
5Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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  • ORCID record for Gary C. Hon
  • For correspondence: Nikhil.Munshi@utsouthwestern.edu Gary.Hon@utsouthwestern.edu
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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.

HIGHLIGHTS

  • Single-cell enhancer perturbation screens during human cardiomyocyte differentiation.

  • Perturbation of CHD-linked enhancers/genes causes deficient CM differentiation.

  • Repression or knockout of TBX5 enhancers delays transition from mid to late CM states.

  • Deficient differentiation coincides with reduced expression of known cardiac genes.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵^ Lead contact

  • 1. We generated and validated H9 hESCs with TBX5 exon deletion, and we performed comparative studies with TBX5 enhancer knockouts during cardiomyocyte differentiation. 2. We performed new experiments to independently phenotype TBX5 enhancer KOs in comparison to TBX5 exon KOs and WT cells. New experiments included: single-cell RNA-Seq, qPCR, immunocytochemistry, and FlowFISH (PrimeFlow). 3. We performed target amplification studies of putative enhancer target genes to verify gene expression changes. 4. To validate the integrity of cell cluster labeling, we performed cell label transfer analysis. 5. We improved the single-cell analysis to give more molecular insights on enhancer perturbations, including downstream target genes.

Copyright 
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 4.0 International license.
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Posted January 20, 2023.
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CHD-associated enhancers shape human cardiomyocyte lineage commitment
Daniel A. Armendariz, Sean C. Goetsch, Anjana Sundarrajan, Sushama Sivakumar, Yihan Wang, Shiqi Xie, Nikhil Munshi, Gary C. Hon
bioRxiv 2022.04.07.487515; doi: https://doi.org/10.1101/2022.04.07.487515
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CHD-associated enhancers shape human cardiomyocyte lineage commitment
Daniel A. Armendariz, Sean C. Goetsch, Anjana Sundarrajan, Sushama Sivakumar, Yihan Wang, Shiqi Xie, Nikhil Munshi, Gary C. Hon
bioRxiv 2022.04.07.487515; doi: https://doi.org/10.1101/2022.04.07.487515

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