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The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming

Abstract

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1,2,3,4,5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

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Fig. 1: PHF7 promotes direct cardiac reprogramming.
Fig. 2: PHF7 globally activates the cardiac transcriptome.
Fig. 3: PHF7 reprogrammes cells to a cardiac fate in the absence of exogenous Gata4.
Fig. 4: PHF7 binds to and activates cardiac enhancers.
Fig. 5: PHF7 interacts with SMARCD3 to promote reprogramming.

Data availability

All RNA-seq, ChIP-seq and ATAC-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE151328. Previously published ChIP-seq and single-cell RNA-seq data that were re-analysed here are available under accession codes GSE90893, GSE112315, GSE100471 and http://singlecell.stemcells.cam.ac.uk/mesp1. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The custom R script for plotting the ROSE results have been deposited to GitHub with the access link as https://github.com/chenkn009/PlotROSE.

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Acknowledgements

We thank J. Cabrera for graphical assistance; J. Xu, X. Liu and the Sequencing Core Facility at the Children’s Research Institute and the Genomics and Next Generation Sequencing Core Facility at UT Southwestern for performing the Illumina sequencing. We are grateful to A. Mobley and the Flow Cytometry Core Facility for their assistance. This work was supported by grants from the NIH (HL-130253, HL-138426 and HD-087351), the Foundation Leducq Transatlantic Networks of Excellence in Cardiovascular Research and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). G.A.G. was supported by a NIH T32 Training grant (5T32HL125247-04). H.Z. was supported by a predoctoral fellowship (14PRE20030030) from the American Heart Association. H.H. was supported by a Uehara Memorial Foundation Postdoctoral Fellowship and a Kanae Foreign Study Grant. M.G.M. was a Pew Latin American Fellow in the Biomedical Sciences, supported by the Pew Charitable Trusts.

Author information

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Authors

Contributions

G.A.G., K.C., H.Z. and H.H. designed and performed experiments, data analyses, discussion and writing. S.B. and M.G.M. contributed to experimental work and discussion. N.L. and R.B.-D. contributed to discussion and writing. E.N.O. supervised the study and contributed to discussion and writing.

Corresponding author

Correspondence to Eric N. Olson.

Ethics declarations

Competing interests

E.N.O. is a co-founder and member of the Scientific Advisory Board of Tenaya Therapeutics and holds equity in the company. The other authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Kenneth Chien, Gerald Crabtree and the other, anonymous, reviewer for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 PHF7 augments cardiac reprogramming.

(a) tSNE plots from single-cell RNA Seq analysis of Mesp1+ cardiac progenitors from E6.5-E7.5 demonstrate robust expression of Phf7 and Gata4 throughout the existence of these cells. b, Western blot demonstrating increased cTnT and αMHC-GFP protein expression in day7 AGHMT ±PHF7 TTF iCLMs. Arrow indicates relevant cTnT band. GAPDH is a loading control. Biologically independent experiments were performed with similar results two times. c, Representative FACs plot demonstrating side and forward scatter gating of live cells. Biologically independent experiments were performed three times with similar results across all FACs experiments in this manuscript. d, Quantitative analysis by flow cytometry demonstrates % αMHC-GFP+ and % cTNT+ cells in day 7 AGHMT ±PHF7 TTF iCLMs. n=3 biologically independent samples. p****<0.0001. Data are presented as mean ±SD values. (e) qPCR of TTF iCLMs 7 days after infection with AGHMT ±PHF7 for cardiac markers. Relative to Empty virus control. Normalized to GAPDH. n=2 biologically independent samples. f, Quantitative analysis of immunocytochemistry in Day 21 My-GHMT±PHF7 human CFs. n=4 biologically independent samples. p****<0.0001. Data are presented as mean ±SD values. (g) qPCR of Day 21 My-GHMT±PHF7 human CF iCLMs for cardiac markers. Relative to Empty virus control. Normalized to GAPDH. n=2 biologically independent samples. All statistical comparisons between groups were evaluated by one-way ANOVA analysis, with modification for multiple comparisons. Source data are provided as a Source data file.

Source data

Extended Data Fig. 2 Transient PHF7 expression augments cardiac reprogramming.

a, Schematic for doxycycline-inducible expression strategy of PHF7 in reprogramming. b, Schematic representation of doxycycline (dox) dosing strategy. Dox was administered for different intervals; 4 weeks on (D1 on), 6 days (D3 off), and 13 days (D10 off), immediately following infection of TTFs or MEFs with pRetroX-PHF7/pMXs-AGHMT/pMXs-PHF7. Cardiac reprogramming media was added at D0 and changed along with dox every 2 days. c, Western blot demonstrating activation of PHF7 expression in the presence of dox (100-1000ng/mL). GAPDH is a loading control. Biologically independent experiments were performed with similar results two times. (d) qPCR for PHF7 expression iCLM MEFs in the indicated samples after 28 days. n=3 biologically independent samples, p*=0.0468, p****<0.0001. Data are presented as mean ±SD values. (e) qPCR for Myh6 expression in iCLM TTFs in the indicated samples after 28 days. n=2 biologically independent samples. f, Representative immunocytochemistry for αMHC-GFP (green) and cTNT (red) in indicated treatment groups for Day 28 TTF iCLMs and (g) quantification of %αMHC-GFP+, % cTnT+, and %α-actinin+ cells in indicated treatment groups Hoechst (blue). Scale=100μm. n=3 biologically independent samples. %αMHC-GFP: D1 on p***=0.0001, D3 off p****<0.0001, D10 off p***=0.0007. % cTnT+: D1 on p*=0.0117, D3 off p**=0.0015, D10 off p***=0.001. %α-actinin+: D1 on p***=0.001, D3 off p***=0.0006, D10 off p***= 0.0003. Data are presented as mean ±SD values. h, Immunocytochemistry for α-actinin in D3 off TTF iCLMs at day 28. Zoomed view demonstrates sarcomere organization. Scale=100μm. Biologically independent experiments were performed with similar results three times. i, Quantification of number of spontaneously beating cells per high power field in MEF iCLMs. n=3 biologically independent experiments. Day 7: D1 on p*** 0.0007, D3 off p****<0.0001, Day 10 off p**=0.0028. Day 10: D1 on p**=0.0071, D3 off p***=0.0009, D10 off p**=0.0041. Day 21: D1 on p***=0.0006, D3 off p****<0.0001, D10 off p***=0.002. j, Quantification of intracellular calcium flux by Fluo-4 assay in day 28 iCLM MEFs. n=3 biologically independent samples. p****<0.0001. Data are presented as mean ±SD values. All statistical comparisons between groups were evaluated by one-way ANOVA analysis, with modification for multiple comparisons. Source data are provided as a Source data file.

Source data

Extended Data Fig. 3 PHF7 activates the cardiac transcriptome.

a, Unbiased heatmap displaying the 50 most upregulated transcripts by RNA-Seq in the presence of AGHMT+PHF7. b, Unbiased heatmap displaying the 50 most downregulated transcripts by RNA-Seq in the presence of AGHMT+PHF7. Color scale by Z score. c, Plots of cardiac reprogramming transcription factors upregulated in the presence of PHF7 by RNA-Seq analyses (normalized counts per million). n=3 biologically independent replicates. p****<0.0001. Data are presented as mean ±SD values. d, Western blot of lysates from day 7 TTF iCLMs following GHMT and AGHMT reprogramming ±PHF7 blotting for Gata4 and Mef2c expression. GAPDH is a loading control. Biologically independent experiments were performed with similar results two times. e, RNA-seq tracks at Gata4 3’ UTR and Tbx5 3’UTR from Empty, AGHMT (5F), and AGHMT+PHF7 (5F+PHF7) samples. f, g, Enrichment plots of indicated gene sets and their nominal p-value of genes upregulated by PHF7 (f) and downregulated by PHF7 (g). h, EdU pulse labeling of day 1 TTF iCLMs in the presence of Empty, PHF7, or AGHMT±PHF7. EdU (magenta), Hoechst (blue). Scale=100μm. Biologically independent experiments were performed with similar results three times. (i) Quantification of EdU pulse labeling of day 1 TTF iCLMs in the presence of Empty, PHF7, or AGHMT±PHF7. n=3 biologically independent samples. Empty v AGHMT: p***0.0008. AGHMT±PHF7: ns=0.9993. Data are presented as mean ±SD values. All statistical comparisons between groups were evaluated by one-way ANOVA analysis, with modification for multiple comparisons. Source data are provided as a Source data file.

Source data

Extended Data Fig. 4 PHF7 augments reprogramming with GMT, GHMT, and AGHMT.

a, Representative immunocytochemistry images demonstrate that PHF7 augments GMT, GHMT, or AGHMT reprogramming in both αMHC-GFP transgenic TTF and MEF iCLMs at day 7. αMHC-GFP (green), scale=200μm. Biologically independent experiments were performed with similar results three times. b, Quantitative analysis of % of αMHC-GFP+, cTnT+, and double positive TTF iCLMs 7d after infection with MT, GMT, GHMT, or AGHMT ±PHF7. n=2 biologically independent samples. c, Quantification number of cells with active calcium flux per high-powered field by Fluo-4 assay in Day 14 iCLM MEFs infected with GMT, GHMT, or AGHMT ±PHF7. n=3 biologically independent samples, p***=0.0003. Data are presented as mean ±SD values. All statistical comparisons between groups were evaluated by one-way ANOVA analysis, with modification for multiple comparisons. Source data are provided as a Source data file.

Source data

Extended Data Fig. 5 PHF7 binds to cardiac enhancers.

a, Western blot for PHF7 of lysates from HEK293 cells transfected with PHF7, N-terminal 3x-Ty1-PHF7, or C-terminal PHF7-3xTy1. GAPDH is a loading control Biologically independent experiments were performed with similar results two times. b, Representative immunocytochemistry images demonstrate that PHF3xTy1 augments reprogramming to a similar extent as untagged PHF7 in day7 MEF iCLMs. Scale=200μm. Biologically independent experiments were performed with similar results three times. c, Genomic location of annotated PHF73xTy1 peaks in the presence of reprogramming factors (AGHMT+ PHF73xTy1 -unique). d, Genomic location of annotated PHF73xTy1 peaks in the absence of reprogramming factors (PHF73xTy1 -unique). e, GO enrichment analysis of PHF73xTy1 peaks in the absence of AGHMT (PHF73xTy1 -unique) as determined by GREAT analysis. f, GO enrichment analysis of PHF73xTy1 peaks in the presence of AGHMT (AGHMT+ PHF73xTy1 -unique) as determined by GREAT analysis. (g) de novo motif analysis of PHF73xTy1 peaks in the absence of AGHMT by HOMER (PHF73xTy1 -unique). (FDR threshold 10−3). h, Motif frequency analysis of Gata4, Mef2c, Hand2, Tbx5, TEAD, and CTCF centered on PHF7 peak signal in reprogramming (AGHMT+PHF73xTy1)-unique peaks (red), unchanged PHF73xTy1 peaks (gray), or PHF73xTy1-unique peaks in the absence of reprogramming factors (green). i, Heatmap ordered by PHF73xTy1 signal in absence of AGHMT (PHF73xTy1 -unique peaks) aligned with H3K27ac, Gata4, Hand2, Mef2c, Tbx5 ChIP signal from day2 AGHMT iCLMs. Normalized ChIP-seq signal with ±2kb window centered around peak summit and sorted in descending order by signal intensity. Source data are provided as a Source data file.

Source data

Extended Data Fig. 6 PHF7 aligns with H3K4me2 and other active histone marks.

a, Heatmap ordered by PHF73xTy1 peaks aligned with H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K79me2, and H3K27me3 histone ChIP from uninduced MEFs. Normalized ChIP-seq signal with ±2kb window centered around peak summit and sorted in descending order by signal intensity. b, Heatmap ordered by PHF73xTy1 peaks bound to P4 heart enhancers aligned with H3K4me1, H3K4me2, H3K4me3, H3K27ac, H3K79me2, and H3K27me3 histone ChIP from uninduced MEFs. Normalized ChIP-seq signal with ±2kb window centered around peak summit and sorted in descending order by signal intensity.

Extended Data Fig. 7 PHF7 activates cardiac enhancers.

a, Schematic of enhancer-hsp68-mCherry retroviral generation and application to AGHMT±PHF7 MEF iCLM reprogramming. b, Representative images of Myh6enh, Phf7enh, Thraenh, Tns1enh, and Tbx20enh-hsp68-mCherry activation in Day 4 AGHMT±PHF7 MEF iCLMs. mCherry (red), Hoechst (blue). Scale=100μm. Biologically independent experiments were performed with similar results three times. c, Quantification of enhancer activation by %mCherry+ cells in Day 4 AGHMT±PHF7 MEF iCLMs treated with respective enhancer-hsp68-mCherry constructs. n=3 biologically independent samples. Myh6enh: p***=0.0006, Phf7enh: p****<0.0001, Thraenh: p***=0.0001, Tns1enh: p**=0.0023, Tbx20enh: p***=0.0008. Data are presented as mean ±SD values. All statistical comparisons between groups were evaluated by one-way ANOVA analysis, with modification for multiple comparisons. Source data are provided as a Source data file.

Source data

Extended Data Fig. 8 PHF7 participates in a core TF autoregulatory circuit.

a-f, Genome browser shots of PHF73xTy1 binding ±AGHMT at the a) Tnni1/Tnnt2, b) Gata4, (c) Hand2, (d) Mef2c, (e) Tbx5, and (f) Phf7 super-enhancer loci, aligned with binding profiles of cardiac TFs by Gata4, Hand2, Mef2c, Tbx5, and H3K27ac ChIP in day 2 AGHMT iCLMs. g, Genome browser shot of P4 mouse atrium and ventricle TF (Gata4, Nkx2-5, and Tbx5) and H3K27ac ChIP alignment at the Phf7 locus. h, FLAG co-immunoprecipitation in HEK293 cells transfected with PHF7FLAG and GFP, Gata4myc, Hand2myc, or Mef2cmyc. (IP) Immunoprecipitation. (IB) Immunoblot. GAPDH is a loading control. Biologically independent experiments were performed with similar results three times. i, PHF73xTy1 ChIP qPCR in day2 iCLMs at the Gata4 locus treated with PMT as compared to IgG control. n=3 biologically independent samples, p*=0.0261, by unpaired two-tailed Student’s t-test. Data are presented as mean ±SEM values. Mef2c3xTy1 ChIP qPCR at the Gata4 locus in day 2 iCLMs treated with Mef2c3xTy1+Tbx5 (MT) or PHF7+Mef2c3xTy1+Tbx5 (PMT) as compared to IgG control. n=3 biologically independent replicates. p*=0.0250, by one-way ANOVA with adjustment for multiple comparisons. Data are presented as mean ±SD values. H3K27ac ChIP qPCR at the Gata4 locus in day2 iCLMs treated with Empty vector, MT, or PMT cocktail. n=3 biologically independent samples. p*=0.0320. Data are presented as mean ±SD values. j, PHF73xTy1 ChIP qPCR in day2 iCLMs at the Myh6 locus treated with PMT as compared to IgG control. n=3 biologically independent samples. p*=0.0184, as determined by unpaired two-tailed Student’s t test. Mef2c3xTy1 ChIP qPCR at the Myh6 locus in day 2 iCLMs treated with Mef2c3xTy1+Tbx5 (MT) or PHF7+Mef2c3xTy1+Tbx5 (PMT) as compared to IgG control. n=3 biologically independent samples. p*=0.0118, as determined by one-way ANOVA with adjustment for multiple comparisons. H3K27ac ChIP qPCR at the Myh6 locus in day2 iCLMs treated with Empty vector, MT, or PMT cocktails. n=3 biologically independent samples. p*=0.0202, as determined by one-way ANOVA with adjustment for multiple comparisons. Data are presented as mean ±SD values. Source data are provided as a Source data file.

Source data

Extended Data Fig. 9 PHF7 interacts with cardiac proteins in reprogramming.

a, Table with top shared proteomics hits ranked based on fold change relative to control and normalized abundance from miniTurbo proximity biotinylation assay performed in PHF7miniTurbo and AGHMT+PHF7miniTurbo infected MEFs. b, Heatmap demonstrating AGHMT+PHF7miniTurbo -unique proteins (related to Supplementary Data Table 2). Biologically independent experiments for AGHMT+PHF7miniTurbo samples were performed with similar results two times. c, GO Pathway analysis of unique and shared proteins between PHF7miniTurbo and AGHMT+PHF7miniTurbo biotin-treated samples. d, Streptavidin IP of AGHMT(5F)+PHF7miniTurbo infected MEFs in biotin-treated and untreated control followed by western blot. (IP) Immunoprecipitation. (IB) Immunoblot. Biologically independent experiments were performed with similar results two times. e, Immunocytochemistry images demonstrate overexpression of pMXs-SMARCD3 with AGHMT or AGHMT+PHF7 does not augment reprogramming in day 7 TTF iCLMs. αMHC-GFP (green), Hoechst (blue). Biologically independent experiments were performed with similar results three times. f, Realtime PCR validation of Smarcd3 transcript knockdown by shSmarcd3 in murine neuro2a (N2a). n=2 biologically independent samples. Source data are provided as a Source data file.

Source data

Supplementary information

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Supplementary Video 1

Spontaneously beating AGHMT (5F) ± PHF7 MEF iCLMs at day 10.

Supplementary Video 2

Spontaneously beating AGHMT (5F) ± PHF7 MEF iCLMs at day 27.

Supplementary Video 3

Fluo-4 visualization assay of spontaneous intracellular calcium flux in AGHMT (5F) ± PHF7 MEF iCLMs at day 14.

Supplementary Video 4

Spontaneously beating no dox and D3-off MEF iCLMs at day 21.

Supplementary Video 5

Fluo-4 visualization assay of spontaneous intracellular calcium flux in PMT-treated MEF iCLMs at day 14.

Supplementary Table 1–3

Supplementary Table 1: Annotation and rank of in vivo cardiac SE elements as defined by H3K27ac ChIP signal in P4 mouse ventricle (Table 1a). Overlay of PHF7 ChIP (PHF73xTy1 and 5F + PHF73xTy1) signal at in vivo P4 cardiac SE elements (Table 1b). Supplementary Table 2: Normalized proteomics mass spectrometry data for empty vector, PHF7-mTurbo and AGHMT+PHF7-mTurbo. Supplementary Table 3: Genomic coordinates for enhancers cloned into enhancer–hsp68–mCherry constructs.

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Garry, G.A., Bezprozvannaya, S., Chen, K. et al. The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming. Nat Cell Biol 23, 467–475 (2021). https://doi.org/10.1038/s41556-021-00668-z

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