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A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation

Nature Cardiovascular Research volume 3, pages 317–331 (2024)Cite this article

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Abstract

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.

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Fig. 1: A pooled CRISPR screen identifies BRD4 as a regulator of cardiogenesis.
Fig. 2: Brd4 deletion in ISL1+ progenitors in vivo results in myocardial hypoplasia.
Fig. 3: Brd4 deletion in SHF progenitors in vivo recapitulates loss in ISL1+ progenitors.
Fig. 4: Loss of BRD4 in vivo attenuates cardiac differentiation-related gene expression.
Fig. 5: BRD4 is enriched at transcriptionally active regions of chromatin marked by H3K4Me3.
Fig. 6: BRD4 specifically regulates an MSX1/2+ progenitor population.

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Data availability

All data supporting the findings of this study are included in the main article and associated files, and Source Data have been provided with this manuscript. All transcriptomic and epigenomic data are available in the Gene Expression Omnibus database under accession number GSE184922, which is available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184922.

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Acknowledgements

The authors thank the Srivastava and Jain laboratories for critical discussions and feedback and K. Ozato (National Institutes of Health) for experimental reagents. We are grateful to V. Vedantham (University of California, San Francisco (UCSF)), S. Hota (Gladstone Institutes), I. Kathiriya (UCSF) and M. Costa (Gladstone Institutes) for thoughtful commentary on the manuscript. We thank B. Taylor (Gladstone Institutes) and K. Claiborn (Gladstone Institutes) for editorial assistance as well as A. Silva (Ana Silva Illustrations) and G. Maki (Gladstone Institutes) for assistance with illustrations. We thank the University of Pennsylvania iPSC core for technical assistance. This work was supported by the NIH (R35 HL166663 and R01 HL139783; F31 HL147416 to R.L-S.; K08HL157700 to A.P.; P01 HL146366, R01 HL057181, R01 HL127240 and R01 HL015100 to D.S.), the Burroughs Wellcome Foundation Career Award for Medical Scientists (R.J.), the Allen Foundation (R.J.), the American Heart Association (R.J. and S.M.), the National Science Foundation (15-48571 to R.J.), the Swiss National Science Foundation (P400PM_186704 and P2LAP3_178056 to M.A.), the Japan Society for the Promotion of Science Overseas Research Fellowship (T.N.), the Sarnoff Cardiovascular Research Foundation (A.P.), the Michael Antonov Charitable Foundation (A.P.), the Frank A. Campini Foundation (A.P.), the Tobacco‐Related Disease Research Program (578649 to A.P.), the A. P. Giannini Foundation (P0527061 to A.P.), the Roddenberry Foundation (D.S.), the L. K. Whittier Foundation (D.S.), Dario and Irina Sattui (D.S.) and the Younger Family Fund (D.S.).

Author information

Author notes

  1. Saptarsi M. Haldar

    Present address: Amgen Research, Cardiometabolic Disorders, South San Francisco, CA, USA

Authors and Affiliations

  1. Gladstone Institutes, San Francisco, CA, USA

    Arun Padmanabhan, T. Yvanka de Soysa, Angelo Pelonero, Clara Youngna Lee, Nandhini Sadagopan, Tomohiro Nishino, Lin Ye, Sanjeev S. Ranade, Michael Alexanian, Saptarsi M. Haldar & Deepak Srivastava

  2. Department of Medicine, University of California, San Francisco, School of Medicine, San Francisco, CA, USA

    Arun Padmanabhan, Clara Youngna Lee, Nandhini Sadagopan & Saptarsi M. Haldar

  3. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Arun Padmanabhan

  4. Department of Medicine, University of California, San Diego, School of Medicine, San Diego, CA, USA

    Valerie Sapp & Mohit Jain

  5. Department of Pharmacology, University of California, San Diego, San Diego, CA, USA

    Valerie Sapp & Mohit Jain

  6. Cardiovascular Institute, Epigenetics Institute, and Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Parisha P. Shah, Qiaohong Wang, Li Li, Rachel Yang, Ashley Karnay, Nikhita Bolar, Ricardo Linares-Saldana & Rajan Jain

  7. Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA

    Parisha P. Shah, Qiaohong Wang, Li Li, Rachel Yang, Ashley Karnay, Andrey Poleshko, Nikhita Bolar, Ricardo Linares-Saldana & Rajan Jain

  8. Department of Pediatrics, University of California, San Francisco, School of Medicine, San Francisco, CA, USA

    Michael Alexanian & Deepak Srivastava

  9. Division of Newborn Medicine, Boston Children’s Hospital, Boston, MA, USA

    Sarah U. Morton

  10. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Sarah U. Morton

  11. Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, CA, USA

    Deepak Srivastava

  12. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

    Deepak Srivastava

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  1. Arun Padmanabhan
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Contributions

A.P., R.J. and D.S. conceived and designed the study. A.P., Y.d.S., V.S., P.P.S., Q.W., L.L., C.Y.L., N.S., A. Poleshko, N.B., R.L.-S., T.N. and L.Y. performed all the experiments. A.P., Y.d.S., V.S., A. Pelonero, S.U.M., M.J., R.Y., A.K., L.Y. and R.J. analyzed the data. M.A., S.M.H. and D.S. assisted with data interpretation. A.P. and R.J. wrote the manuscript. D.S. and R.J. supervised the project. All authors edited and approved the manuscript.

Corresponding authors

Correspondence to Arun Padmanabhan, Deepak Srivastava or Rajan Jain.

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Competing interests

D.S. is a scientific co-founder, shareholder and director of Tenaya Therapeutics. S.M.H. is an executive, officer and shareholder of Amgen and is a scientific co-founder and shareholder of Tenaya Therapeutics. M.J. is founder, shareholder and executive of Sapient Bioanalytics, LLC. The remaining authors declare no competing interests.

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Peer review information

Nature Cardiovascular Research thanks Bernice Morrow, John Hinson, Michael A. Burke, Lijie Shi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Integrating a CM differentiation screen and CHD variants.

(a) Top 1000 genes ranked by enrichment or depletion. (b, c) Categorization of top 200 genes enriched (b) or depleted (c) in cardiac myocytes compared to undifferentiated hiPSCs by biological groups. (d, e) Gene ontology (GO) analysis of top enriched (d) or depleted (e) hits (see Methods for details). (f-g) Venn diagrams demonstrating the number of CHD (f) or Non-CHD (g) probands with predicted damaging DNVs in hits identified in our screen as enriching hiPSC:CM differentiation or depleting hiPSC:CM differentiation. Arrows depict Venn diagrams representing the number of probands from each cohort with predicted damaging DNVs identified in our screen that have mutations in known dominant CHD genes or where these candidate CHD genes may potentially be causative. (h) Venn diagram demonstrating the number of CHD probands with (purple) or without (brown) damaging DNVs in hits identified in our screen highlighting no enrichment for extracardiac anomalies (p = 0.43) or neurodevelopmental delay (NDD; p = 0.23) and a slight enrichment for conotruncal CHD (p = 0.04). (i) TNNT2+ cells quantified by flow cytometry at day 10 of hiPSC to CM differentiation in WTC11 cells treated with 100 nM JQ1 starting at day 6 (n = 3 biologically independent samples). (j) MZ3 treatment (500 nM for 0, 3.5, 7, and 16 hours) effectively degrades BRD4 in SV20 hiPSCs as assessed by immunoblot analysis for BRD4 with β-actin expression as a loading control. In all graphs, error bars represent ±1 SEM. * represents p = 0.0271 (two-tailed unpaired t test).

Source data

Extended Data Fig. 2 Inhibition or deletion of BRD4 inhibits mESC cardiac differentiation.

(a) Expression of Myh6, Nkx2-5, and Tnnt2 at day 7 of mESCs cardiac differentiation treated with JQ1 (100 nM) starting day 5 of differentiation (n = 3 biologically independent samples). (b) TNNT2+ cells quantified by flow cytometry at d9 of mESC differentiation in CMV-CreERT2;Brd4flox/flox cells treated with 4-hydroxytamoxifen (TAM), JQ1 (250 nM) or MZ3 (500 nM) starting at day 5 (n = 3 biologically independent samples; FACS gating strategy in Supplementary Fig. 1). (c-d) Immunofluorescence of TNNT2 at day 9 of mESC to CM differentiation in wild type cells treated with JQ1 (100 nM) or vehicle starting at day 5. (e-f) Immunofluorescence of BRD4 in vehicle (ethanol, e-e′′) and TAM (f-f′′) treated undifferentiated mESCs. (g) Volcano plot showing RNA-seq from CMV-CreERT2;Brd4flox/flox (TAM vs. vehicle [VEH]) mESCs and gene ontology analysis of downregulated and upregulated genes (see Methods for details). (h) Heatmap showing expression of select transcription factor and muscle structural protein genes from RNA-seq in CMV-CreERT2;Brd4flox/flox (TAM vs. VEH) mESC-derived cardiac tissues (day 10; TAM or VEH added at day 5). In all graphs, error bars represent ±1 SEM. For a, all comparisons are made relative to 0 nM compound for each gene; * represents p < 0.0493, ** represents p < 0.0085 (two-tailed unpaired t test). For b, all comparisons are made relative to VEH for each condition; * represents p < 0.0188 (two-tailed unpaired t test). Scale Bars = 100 µm (c, d, e, e′, e′′, f, f′, f′′).

Source data

Extended Data Fig. 3 Loss of BRD4 in Isl1Cre-SHF progenitors in vivo results in myocardial hypoplasia.

(a) Lineage tracing of Isl1Cre/+;Brd4flox/+ and Isl1Cre/+;Brd4flox/flox with R26mTmG/+ allele. Immunohistochemistry of ISL1-derived cells (GFP) and TNNT2 or BRD4 (red) in heart and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes at E14 (5 sections from n = 2 control embryos and 11 sections from n = 4 mutant embryos). (b) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+;Brd4flox/+ and Isl1Cre/+;Brd4flox/flox embryos at E12.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (10 sections from n = 2 control embryos and 11 sections from n = 2 mutant embryos). (c) Quantification of percentage phospho-histone H3-, cleaved caspase 3-, and TUNEL-positive cells in the RV of E12.5 and E14.5 embryos of indicated genotypes (n = 2 biologically independent samples per genotype at E12.5; n = 3-4 biologically independent samples per genotype at E14.5). (d) Hematoxylin and eosin staining of a section through outflow tract and RV of Isl1Cre/+;Brd4flox/+ and Isl1Cre/+;Brd4flox/flox embryos at E10 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (9 sections from n = 3 control embryos and 8 sections from n = 3 mutant embryos). (e) Quantification of percentage phospho-histone H3- and TUNEL-positive cells in the RV of E10 control (Isl1Cre/+;Brd4flox/+ or Brd4flox/flox) and Isl1Cre/+;Brd4flox/flox embryos (n = 4 biologically independent samples per condition). (f) BRD4 and TNNT2 immunohistochemistry along with lineage tracing with R26mTmG/+ allele in Isl1Cre/+;Brd4flox/+ and Isl1Cre/+;Brd4flox/flox E9.5 embryos at level of right ventricle. Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p = 0.0091, ** represents p = 0.0021, **** represents p < 0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle; OT, outflow tract. Scale Bars = 100 µm (a, b, d, f).

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Extended Data Fig. 4 Loss of BRD4 in Mef2c-AHF-Cre-SHF CPCs in vivo results in right ventricular thinning.

(a) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre;Brd4flox/+ and Mef2c-AHF-Cre;Brd4flox/flox embryos at E13.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (19 sections from n = 3 control embryos and 18 sections from n = 3 mutant embryos). (b) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E13.5 Mef2c-AHF-Cre;Brd4flox/+ and Mef2c-AHF-Cre;Brd4flox/flox embryos (n = 3-4 biologically independent samples per genotype). (c) Hematoxylin and eosin staining of a section through outflow tract and RV of Mef2c-AHF-Cre;Brd4flox/+ and Mef2c-AHF-Cre;Brd4flox/flox embryos at E10.5 and quantitative assessment of right ventricular myocardial wall thickness in indicated genotypes (6 sections from n = 3 control embryos and 6 sections from n = 3 mutant embryos). (d) Quantification of percentage phospho-histone H3- and cleaved caspase 3-positive cells in the RV of E10.5 Mef2c-AHF-Cre;Brd4flox/+ and Mef2c-AHF-Cre;Brd4flox/flox embryos (n = 3 biologically independent samples per genotype). Error bars represent ±1 SEM. All comparisons are made as indicated; * represents p = 0.0489, ** represents p = 0.0146, **** represents p < 0.0001 (two-tailed unpaired t test). RV, right ventricle; LV, left ventricle. Scale Bars = 100 µm (a, c).

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Extended Data Fig. 5 Wnt signaling is dysregulated upon BRD4 depletion in CPCs.

(a) Image of Brd4flox/flox embryo appearing in Fig. 2i with region microdissected for bulk RNA-seq highlighted in red. (b) Principal component analysis of RNA-seq from Isl1Cre/+;Brd4flox/+ (blue) and Isl1Cre/+;Brd4flox/flox (red) E9.5 embryos. (c) Volcano plots of E9.5 Isl1Cre/+;Brd4flox/+ vs. Isl1Cre/+;Brd4flox/flox embryonic hearts (same data appearing in Fig. 4) with a subset of cardiac and Wnt-related genes annotated (see Methods for details). Brd4flox/flox(d, f) and Isl1Cre/+;Brd4flox/flox (e, g) E9.5 embryos at level of right ventricle stained with ISL1 (red, d-e), BRD4 (green, d-e), or AXIN2 (green, f-g). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the RV from distal outflow tract in mutant embryos. (h-m) ISL1 and AXIN2 Immunohistochemistry of E10.5 Mef2c-AHF-Cre;Brd4flox/+ (h,k) and Mef2c-AHF-Cre;Brd4flox/flox (i,j,l,m) embryos at the level of outflow tract. (h-j, ISL1; k-m, AXIN2). Note the expansion of ISL1- (arrow heads) and AXIN2- (dotted line) expressing cells into the right ventricle from distal outflow tract in mutant embryos. (n-o) AXIN2 RNAscope of Brd4flox/flox (n,n′) and Isl1Cre/+;Brd4flox/flox (o,o′) E9.5 embryos at the level of the right ventricle (n′ and o′ are magnified images of n and o, respectively). (p-q) ISL1 representative immunofluorescence at day 8 of mESC-derived cardiac cultures treated with vehicle (DMSO; VEH) (p) or JQ1 (500 nM) (q) starting at day 5. (r) Isl1 expression in day 8-9 mESC-derived cardiac cultures treated with increasing doses of JQ1 (0–500 nM; JQ1 added at day 5; n = 3 n = 3 biologically independent samples per dose). (s-t) AXIN2 RNAscope of Mef2c-AHF-Cre;Brd4flox/+ (s,s′) and Mef2c-AHF-Cre;Brd4flox/flox (t,t′) E10.5 embryos at level of right ventricle (s′ and t′ are magnified images of s and t, respectively). For r, all comparisons are made relative to 0 nM compound. *** represents p = 0.0007 (two-tailed unpaired t test). RV, right ventricle; OT, outflow tract. Scale Bar = 250 µm (a), 50 µm (d-m, n, o, s, t), 100 µm (p, q, n′, o′, s′, t′).

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Extended Data Fig. 6 BRD4 regulates Wnt signaling during CM differentiation.

(a) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2;Brd4flox/flox mESCs partially normalizes expression of CPC markers and Msx1/2 by qRT-PCR (n = 3-4 biologically independent samples per genotype). (b-e) Attenuating Wnt signaling at the CPC stage (day 5) in mESC to CM differentiation by doubling the normal concentration of the small molecule Wnt inhibitor XAV939 concomitant with Brd4 genetic deletion by 4-hydroxytamoxifen treatment (TAM) in CMV-CreERT2;Brd4flox/flox mESCs partially normalizes TNNT2 staining by immunofluorescence at day 9 of CM differentiation (for e, n = 3 biologically independent samples per condition). (f, g) Attenuation of Wnt signaling at the CPC stage (day 6) in hiPSC to CM differentiation by addition of the small molecule Wnt inhibitor IWP4 (5 μM for low dose and 10 μM high dose) concomitant with BET inhibition using JQ1 (25 nM for low dose and 50 nM for high dose) increases the number of TNNT2+ cells as assessed by flow cytometry (n = 3 biologically independent samples per condition; gating strategy in Supplementary Fig. 2). Error bars represent ±1 SEM. For a,e,f,g all comparisons are made with p values as indicated (two-way ANOVA with Tukey’s multiple comparisons test).

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Extended Data Fig. 7 Generation of BRD4FLAG/FLAG hiPSC line and BRD4 occupancy in cardiac progenitor cells.

(a) Targeting strategy to introduce 3XFLAG epitope tag into the N-terminus of the endogenous BRD4 locus. (b) Karyotyping results of BRD4FLAG/FLAG hiPSC line. (c) Western blot analysis of protein lysates collected from BRD4FLAG/FLAG hiPSCs using FLAG antibody demonstrates expression of 3XFLAG-tagged BRD4 isoforms that are degraded upon addition of the PROTAC BET degrader dBET650. (d) Pearson correlation matrices demonstrating high reproducibility between replicate CUT&RUN datasets. (e-g) Track view of indicated loci showing CUT&RUN factor occupancy (FLAG, BRD4, H3K4Me3) or H3K27Ac ChIP-seq enrichment in hiPSC-derived CPCs.

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Extended Data Fig. 8 Iterative filtering steps for selection of cells analyzed in scRNA-seq.

(a-c) UMAP plots of all cells (n = 23,592) collected from the microdissected heart and surrounding pharyngeal mesoderm (n = 2 embryos per genotype) (a), labeled by sample identity (b), and number of features (c). (d) Feature plots for expression of example marker genes used to define cell types (for example, Hbb-y for blood cells; Dlx2, Dlx5, and Twist1 for neural crest cells; Lhx2 and Foxc2 for branchiomeric muscle progenitors; Epcam for endoderm). (e-g) UMAP plots demonstrate expression of the Cre transgene (e) occurs in clusters marked by high Mef2c (f) and Isl1 (g) expression, consistent with Cre driven in Mef2c-expressing second heart field cells. (h) Clusters of Cre expressing cells detected at E9.5 in our scRNA-seq dataset selected for further analysis (n = 4,640). (i) Feature plot for Cre expression in a UMAP of cells from (h) following normalization and reclustering. (h’-i’) Cluster 0 cells highlighted in red on UMAP plots from h and i.

Extended Data Fig. 9 Pathway analysis of differentially expressed genes by cluster.

Pathway analysis of differentially expressed genes between mutant (Mef2c-AHF-Cre;Brd4flox/flox) and control (Brd4flox/flox) embryos by cluster for each cellular population identified in our scRNA-seq studies (see Methods for details).

Extended Data Fig. 10 BRD4 loss increases MSX1- and MSX2-positive cells in vivo.

Mef2c-AHF-Cre;Brd4flox/+ (a) and Mef2c-AHF-Cre;Brd4flox/flox (b, c) E10.5 embryos at level of right ventricle stained with MSX1/2 (yellow); inset shows area indicated by arrowheads. RNAscope in Brd4flox/flox (d, e), Isl1Cre/+;Brd4flox/flox (f, g), Mef2c-AHF-Cre;Brd4flox/+ (h, i), and Mef2c-AHF-Cre;Brd4flox/flox (j, k) E9.5-10.5 embryos at level of right ventricle for MSX1 (d,f,h,j) or MSX2 (e,g,i,k). Regions in yellow boxes in d-k are shown in higher magnification in d′-k′. RV, right ventricle; OT, outflow tract. Scale Bars = 50 µm (a-c, d-k), 165 µm (d′, e′, f′, g′), 125 µm (h′, j′), 200 µm (i′, k′).

Supplementary information

Supplementary information

Supplementary Tables 1 and 2, Supplementary Datasets 1 and 2 and Supplementary Figs. 1 and 2

Reporting Summary

Supplementary Tables 1 and 2

Supplementary Dataset 1

Supplementary Dataset 2

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Padmanabhan, A., de Soysa, T.Y., Pelonero, A. et al. A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation. Nat Cardiovasc Res 3, 317–331 (2024). https://doi.org/10.1038/s44161-024-00431-1

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