CHD-associated enhancers shape human cardiomyocyte lineage commitment

  1. Daniel A Armendariz
  2. Sean C Goetsch
  3. Anjana Sundarrajan
  4. Sushama Sivakumar
  5. Yihan Wang
  6. Shiqi Xie
  7. Nikhil V Munshi  Is a corresponding author
  8. Gary C Hon  Is a corresponding author
  1. Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, United States
  2. Department of Internal Medicine, University of Texas Southwestern Medical Center, United States
  3. Division of Cardiology, Department of Molecular Biology, McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, United States
  4. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, United States
  5. Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, United States
6 figures, 1 table and 7 additional files

Figures

Figure 1 with 1 supplement
Single-cell screens of congenital heart defect (CHD)-associated enhancers during cardiomyocyte (CM) differentiation.

(A) Genome browser snapshot emphasizing features of a targeted enhancer about 15 kb upstream of the RBM20 gene. Yellow highlights enhancer region. (B) H3K27ac and open chromatin (ATAC-Seq) …

Figure 1—figure supplement 1
Single-cell CRISPRi screen validation and statistics.

(A) Brightfield images of H9-dCas9-KRAB cells receiving sgRNAs targeting OCT4 or NT control (Scale bar = 200μm). (B) (Top) OCT4 expression in cells receiving sgRNAs targeting OCT4 or NT control. For …

Figure 2 with 1 supplement
CRISPRi of congenital heart defect (CHD)-associated enhancers delays cardiomyocyte (CM) differentiation.

(A) PHATE visualization of CM cells with four Louvain clusters. (B) Feature plot of pseudotime ranking of cells across PHATE trajectory. (C) Feature plot of TNNT2 expression. We defined the top 100 …

Figure 2—figure supplement 1
Single-cell CRISPRi screen cardiomyocyte (CM) subpopulation marker gene expression.

(A) Feature plots of marker genes for SOX4+ progenitors, FN1+ early-stage CMs, ACTA2+ mid-stage CMs, and NPPA+ atrial-like late-stage CMs. (B) Example of sgRNA filtering approach. (Top) p-Values …

Figure 3 with 1 supplement
A focused validation screen demonstrates that TBX5 enhancers modulate cardiomyocyte (CM) cell fate.

(A) (Top) Schematic of validation CRISPRi screen. H9-dCas9-KRAB cells are infected with lentiviral sgRNA library targeting TBX5 enhancers and differentiated over 8 days into CMs followed by …

Figure 3—figure supplement 1
Focused screen marker expression and sgRNA distribution.

(A) Feature plots of marker genes for neuronal (+SOX2), cardiomyocytes (+TNNT2), endoderm (+TTR). (B) Feature plots of cells receiving sgRNAs targeting TBX5 enhancers (red) or non-genome targeting …

Figure 4 with 1 supplement
TBX5 enhancer repression alters cardiomyocyte (CM) molecular signatures.

PHATE visualization of CMs in the focused screen with three Louvain clusters. (B) Feature plot of pseudotime ranking of cells across PHATE trajectory. (C) Feature plot of NPPA expression. (D) …

Figure 4—figure supplement 1
Focused screen cardiomyocyte (CM) subpopulation marker expression and sgRNA distribution.

(A) Feature plots of marker genes for CM progenitors (FN1+), mid-CM (ACTA2+), and late-CM (NPPA+). (B) Feature plots of cells receiving sgRNAs targeting TBX5 enhancers (red) or non-genome targeting …

Figure 5 with 1 supplement
TBX5 enhancer knockouts recapitulate CRISPRi phenotypes.

(A) (Top) TBX5 enhancer knockout strategy. (Bottom) Genome browser snapshots of chromatin status and sequence conservation. Dotted lines denote sgRNA target sites. (B) Genotyping PCR to verify TBX5 …

Figure 5—source data 1

Original genotyping gels for panel B.

Enhancer 3 KO genotyping gel, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data1-v2.zip
Figure 5—source data 2

Original genotyping gels for panel B.

Enhancer 3 KO genotyping gel, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data2-v2.zip
Figure 5—source data 3

Original genotyping gels for panel B.

Enhancer 5 KO genotyping gel, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data3-v2.zip
Figure 5—source data 4

Original genotyping gels for panel B.

Enhancer 5 KO genotyping gel, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data4-v2.zip
Figure 5—source data 5

Original genotyping gels for panel B.

TBX5 exon 3 KO genotyping gel, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data5-v2.zip
Figure 5—source data 6

Original genotyping gels for panel B.

TBX5 exon 3 KO genotyping gel, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data6-v2.zip
Figure 5—source data 7

Original western blots for panel N. Western blot of actin control, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data7-v2.zip
Figure 5—source data 8

Original western blots for panel N. Western blot of actin control, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data8-v2.zip
Figure 5—source data 9

Original western blots for panel N. Western blot of FN1, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data9-v2.zip
Figure 5—source data 10

Original western blots for panel N. Western blot of FN1, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig5-data10-v2.zip
Figure 5—figure supplement 1
TBX5 enhancer knockout cell distribution.

(A) UMAP visualization of H9-derived cells after 8 days of differentiation. Five Louvain clusters indicated. (B) UMAP distribution of TBX5 exon KO and enhancer KO cells after cardiomyocyte (CM) …

Figure 6 with 1 supplement
Heterozygous TBX5 enhancer 5 knockout (KO) displays reduced phenotypes.

(A) TBX5 enhancer 5 heterozygous KO strategy. (B) (Top) Position of primers used to validate TBX5 enhancer 5 heterozygous clone. Red: KO enhancer-spanning primers; blue: left junction primers; …

Figure 6—source data 1

Original genotyping gels for panel B. Enhancer 5 HET genotyping, with labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig6-data1-v2.zip
Figure 6—source data 2

Original genotyping gels for panel B. Enhancer 5 HET genotyping, without labels.

https://cdn.elifesciences.org/articles/86206/elife-86206-fig6-data2-v2.zip
Figure 6—figure supplement 1
TBX5 heterozygous enhancer knockout validation.

(A) Feature plots of marker genes for neuronal (+SOX2), cardiomyocytes (CMs) (+TNNT2), endoderm (+TTR). (B) Feature plots of TBX5 enhancer 3 (orange), enhancer 5 (yellow), and WT (gray) CMs. (C) …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugPuromycinCayman ChemicalCat#13884
Chemical compound, drugBlasticidinRPICat#3513-03-9
Chemical compound, drugThiazovivinSigma-AldrichCat#SML1045
Chemical compound, drugTrypLE SelectThermo FisherCat#12563
Chemical compound, drugCHIR99021TocrisCat#4423
Chemical compound, drugWnt-C59CaymanCat#16644
Chemical compound, drugAccutaseSigma-AldrichCat#SCR005
Chemical compound, drugInsulinGibcoCat#12585014
Chemical compound, drugB-27Thermo FisherCat#17504044
Chemical compound, drugMolecular Probes Fura-2Thermo FisherCat#F1221
Chemical compound, drugPluronic F-127Thermo FisherCat#P6867
AntibodyAnti-human-FN1 (Rabbit polyclonal)Thermo FisherCat#PA5-29578WB(1:1000)
AntibodyAnti-rabbit IgGCell Signaling TechnologyCST #7074WB(1:400)
Chemical compound, drugKAPA HiFi HSKAPACat#KK2502
Commercial assay, kitPrimeFlow RNA Assay KitThermo FisherCat#88-18005-210
Chemical compound, drugNEBNext High-FidelityNew England BiolabsCat#M0541L
Chemical compound, drugGibson Assembly Master MixNew England BiolabsCat#E2611L
Chemical compound, drugmTeSR PlusStemcell TechnologiesCat#100–0276
Chemical compound, drugMatrigelCorningCat#354277
Commercial assay, kitP3 Primary Cell 4D-Nucleofector X KitLonzaCat#V4XP-3024
Commercial assay, kit10× genomics Chromium Single Cell 3′ Kit V3.110× GenomicsCat#PN-1000147
Commercial assay, kit10× CellPlex10× GenomicsCat#PN-1000261
Commercial assay, kit10× Target Hybridization Kit10× GenomicsCat#PN-1000248
Chemical compound, drugRPMI 1640Thermo FisherCat#11875093
Chemical compound, drugKnockOut SerumThermo FisherCat#10828028
Chemical compound, drugHHBSSCorningCat#21-023-CM
Strain, strain background (Endura)Endura ElectroCompetent CellsLucigenCat#60242–2Electrocompetent Cells
Strain, strain background (Escherichia coli)Stellar Competent CellsClontechCat#636766
OtherSingle-cell RNA-seq DataThis paperGEO: GSE190475Sequencing data located at GEO
Cell line (Homo sapiens)293T cellsATCCCRL-3216Mycoplasma free; ATCC STR authenticated as CRL-3216
Cell line (Homo sapiens)H9 cellsWiCellWA09Mycoplasma free; ATCC STR authenticated as WAe009-A-18
Recombinant DNA reagentPlasmid: pMD2.GAddgeneRRID:Addgene_12259
Recombinant DNA reagentPlasmid: psPAX2AddgeneRRID:Addgene_12260
Recombinant DNA reagentPlasmid: lenti-dCas9-KRAB-BlastAddgeneRRID:Addgene_89567
Recombinant DNA reagentPlasmid: CROPseq-Guide-puroAddgeneRRID:Addgene_86708
Sequence-based reagentsgRNA oligosThis papersgRNA oligosSupplementary file 1
Sequence-based reagentqPCR primersThis paperqPCR primersSupplementary file 2
Software, algorithmStarPMID:23104886RRID:SCR_004463
Software, algorithmPicardBroad InstituteRRID:SCR_006525
Software, algorithmFlowCalPMID:27110723RRID:SCR_018140
Software, algorithmFeatureCountsDOI:10.1093/bioinformatics/btt656RRID:SCR_012919
Software, algorithm10× Genomics Cellranger10× GenomicsRRID:SCR_023221
Software, algorithmScanpyPMID:29409532RRID:SCR_018139
Software, algorithmIGVBroad InstituteRRID:SCR_011793
OtherIllumina NextSeq 500 instrumentIlluminaNext-generation sequencer
OtherIllumina NextSeq 2000 instrumentIlluminaNext-generation sequencer
OtherIllumina NovaSeq 6000 instrumentIlluminaNext-generation sequencer
OtherAgilent 2200 TapeStation instrumentAgilentAutomated electrophoresis instrument
OtherQubit Fluorometric Quantitation instrumentThermo FisherFlurometer
OtherEVOS FL Auto Imaging SystemThermo FisherMicroscope

Additional files

Supplementary file 1

List of target enhancers and sequences used in study.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp1-v2.xlsx
Supplementary file 2

Sequencing statistics.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp2-v2.xlsx
Supplementary file 3

Top 100 Louvain cluster defining genes for each dataset.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp3-v2.xlsx
Supplementary file 4

Differentially expressed genes through hypergeometric test.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp4-v2.xlsx
Supplementary file 5

Focus screen mid-CM and late-CM defining genes.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp5-v2.xlsx
Supplementary file 6

Differentially expressed genes between TBX5 enhancer 5 heterozygous KO cells and WT cells, in the late-CM state.

https://cdn.elifesciences.org/articles/86206/elife-86206-supp6-v2.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/86206/elife-86206-mdarchecklist1-v2.docx

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