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ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage

Nature Cell Biology volume 24, pages 672–684 (2022)Cite this article

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Abstract

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

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Fig. 1: ETV2 promotes the endothelial program in both MEFs and EBs.
Fig. 2: ETV2 targets nucleosomes during reprogramming.
Fig. 3: ETV2 recruits BRG1 for chromatin remodelling.
Fig. 4: Phased nucleosomes are established surrounding ETV2 peaks.
Fig. 5: Brg1 knockdown results in a significant decrease in cells expressing FLK1 during reprogramming.
Fig. 6: ETV2 requires BRG1 to activate downstream genes during reprogramming.
Fig. 7: Brg1 conditional KO in ES/EBs.

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Data availability

The scRNA-seq, bulk RNA-seq, ATAC-seq, ChIP-seq and NOMe-seq data of Etv2-induced EB differentiation and MEF reprogramming are deposited at the NCBI Gene Expression Omnibus (GEO) database under accession no. GSE185684 (GSE168521, ChIP-seq of Etv2-induced MEF reprogramming and EB differentiation; GSE168636, ATAC-seq of Etv2-induced MEF reprogramming and EB differentiation; GSE185682, bulk RNA-seq of Etv2-induced EB differentiation; GSE185683, scRNA-seq of Etv2-induced MEFs reprogramming). All data will be made available upon request. All unique materials used in these studies are readily available from the authors or from commercial sources (Supplementary Tables 3–5). The MEF MNase-seq is from GSE90893. The MEF histone modification ChIP-seq of H3K9me3, H3K27me3, H3K9ac, H3K4me2, H3K4me1 and HDAC1 is from GSE90893. The H3K27ac ChIP-seq in Brg1 KD EB is from GSE71509. The scRNA-seq of iPSC reprogramming is from GSE100344. The scRNA-seq of cardiac fibroblast reprogramming is from GSE98567. The scRNA-seq of neural reprogramming is from GSE67310. The mass spectrometry data are available from GitHub (https://github.com/gongx030/Etv2_pioneer). Source data are provided with this paper.

Code availability

Codes pertaining to important analyses in this study are available from GitHub (https://github.com/gongx030/Etv2_pioneer).

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Acknowledgements

This work was supported by a grant from the Department of Defense (W81XWH2110606) and Minnesota Regenerative Medicine. We thank N. Koyano, K.-D. Choi and B. N. Singh for technical assistance and discussions. We acknowledge G. R. Crabtree and B. Bruneau for providing the Brg1f/f;ActinCreER ES cells. We acknowledge the University of Minnesota Genomics Center for their technical assistance and the Minnesota Supercomputing Institute for providing computational resources. We recognize C. Faraday for assistance with figure layout and preparation.

Author information

Author notes

  1. These authors contributed equally: Wuming Gong, Satyabrata Das, Javier E. Sierra-Pagan.

Authors and Affiliations

  1. Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, USA

    Wuming Gong, Satyabrata Das, Javier E. Sierra-Pagan, Erik Skie, Nikita Dsouza, Thijs A. Larson, Mary G. Garry & Daniel J. Garry

  2. Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA

    Mary G. Garry & Daniel J. Garry

  3. Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA

    Mary G. Garry & Daniel J. Garry

  4. Institute for Regenerative Medicine, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Edgar Luzete-Monteiro & Kenneth S. Zaret

  5. Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA

    Edgar Luzete-Monteiro

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  1. Wuming Gong
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Contributions

W.G., S.D., J.E.S.-P. and D.J.G. conceived the project and W.G., S.D., J.E.S.-P., K.S.Z., M.G.G. and D.J.G. wrote the manuscript. W.G., S.D., J.E.S.-P., E.S., N.D., T.A.L. and E.L.-M., designed, performed experiments and analysed the data. K.S.Z., M.G.G. and D.J.G. supervised the project. All authors commented on and edited the final version of the paper.

Corresponding author

Correspondence to Daniel J. Garry.

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D.J.G. and M.G.G. are co-founders of NorthStar Genomics. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Characterization of mouse embryonic fibroblasts that overexpress ETV2 (iHA-Etv2 MEFs) following the addition of doxycycline to reprogram MEFs to endothelial cells.

(a) The gating strategy for iHA-Etv2 MEF FACS characterization is outlined in the five profiles. (b) We isolated embryonic fibroblasts from this mouse line and demonstrated that this cell population uniformly expressed fibroblast markers (Thy1.2, CD44 and CD29). (c) The western blot analysis showed that ETV2 was robustly expressed within 3 hrs post-Dox treatment [representative blots (c) from 3 independent experiments with similar results]. (d-g) ETV2 overexpression resulted in an increase in cells expressing FLK1/TIE2, as measured by FACS (n = 3 biological replicates; *P < 0.05). (h) The biological processes that are significantly associated with the up-regulated genes in cluster 7 (FLK1+ cells at day 7 of reprogramming) compared with cluster 1 (undifferentiated MEFs). (I-m) qPCR experiments showed the increased expression levels of endothelial genes (i) Etv2 (*P = 0.0423, *P = 0.0377, ****P < 1×10-4), (j) Lmo2 (*P = 0.0496), (k) Flk1 (****P < 1×10-4), (l) CD31 (**P = 0.0044) and (m) Tie2 (****P < 1×10-4) at 1 day, 2 days, and 7 days post induction of ETV2, as well as the no Dox control (n = 3 biological replicates; one-way ANOVA with multiple comparison). Data are presented as mean ± SEM.

Source data

Extended Data Fig. 2 Expression of endothelial transcripts in the FLK1+ cell population at day 7 post-ETV2 induction during MEF reprogramming.

(a) The violin plots show the scaled expression levels of endothelial markers such as Etv2, Emcn, Lmo2, Flk1/Kdr, Cdh5 and Sox18 in MEFs, day 1, day 2, day 7 post-ETV2 induction, as well as the FLK1+ cells from day 7. The y-axis indicates the gene expression levels scaled and normalized by Seurat. (b) The violin plots show the scaled expression levels of endothelial markers in seven cell clusters. The y-axis indicates the gene expression levels scaled and normalized by Seurat. The one-sided enrichment test was used to evaluate the significance of pathway enrichment. The p-value adjustment was performed by B-H Procedure. (c) The biological processes that are significantly associated with the up-regulated in genes in cluster 1 (undifferentiated MEFs) compared with the rest of the cell populations. There are 3,562, 948, 2,936, 7,202 and 827 single cells from undifferentiated MEFs, MEFs with day 1, day 2, day 7 post-ETV2 induction, and FLK1+ cell population of day 7 post-ETV2 induction, respectively. (d) GSEA plot indicates significant upregulation of the inflammatory response in MEFs (cluster 1). The y-axis representing the enrichment score (ES) for each gene and x-axis indicates the gene rank in the ordered list. The default GSEA permutation test was used to evaluate the significance of gene set enrichment. No p-value adjustment was performed. (e) Heatmap representing the gene expression levels scaled by Seurat for upregulated (red) and downregulated (blue) genes in cluster 1 and cluster 2. (f-g) The bar plots show top 10 significant pathways for cluster 1 and cluster 2 for MEFs. The y-axis represents the p adjusted values obtained from over-representation test in Gene Ontology enrichment analysis. The one-sided enrichment test was used to evaluate the significance of Gene Ontology enrichment. The p-value adjustment was performed by B-H Procedure.

Extended Data Fig. 3 Expression profile of immune response related genes and significance of pathways in reprogrammed MEFs are upregulated post induction of ETV2.

(a) The UMAP shows expression profiles of Tlr3, Nfkb1 and Vav3, Cd38 and Abl1 (members of B cell receptor signaling pathway) in undifferentiated MEFs and post Etv2 induction day 1, day 2, day 7 and Flk1+ cells at day 7. (b-c) The bar plot shows immune response related pathways significantly upregulated in (b) Flk1+ cells from day 7 and (c) day 1 post Etv2 induction compared to MEFs. The y-axis indicates the p-value showing significance for each pathway obtained from Gene Ontology enrichment. The one-sided enrichment test was used to evaluate the significance of Gene Ontology enrichment. The p-value adjustment was performed by B-H Procedure.

Extended Data Fig. 4 ETV2 overexpression promotes endothelial lineage development in iHA-Etv2 ES/EBs.

(a) Etv2 endogenous expression during differentiation of ES/EBs (n = 3 biological replicates; one-way ANOVA with multiple comparison ***P = 2×10-4). (b-c) FACS analysis of iHA-Etv2 EBs after 3 h (D2.125) or 12 h (D2.5) of the overexpression of ETV2 ( + Dox). Note a significant induction of the endothelial lineage (FLK1+ cells) following 12 h of ETV2 overexpression (n = 3 biological replicates; 2way ANOVA with multiple comparison ****P < 1×10-4). (d-h) qPCR experiments showed increased expression levels of endothelial genes (d) Etv2 (****P < 1×10-4, ***P = 0.0004), (e) Cdh5 (***P = 0.0001), (f) Lmo2 (*P = 0.041, ****P < 1×10-4), (g) Flk1 (***P = 0.0002, ****P < 1×10-4) and (h) Sox18 (***P = 0.0005) following the induction of ETV2 ( + Dox). (n = 3 biological replicates; one-way ANOVA with multiple comparison). Data are presented as mean ± SEM.

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Extended Data Fig. 5 Commonly up- and down-regulated genes in FLK1 + cell populations from ETV2 induced ES/EB differentiation and MEF reprogramming.

(a-b) The Venn diagrams show the overlap of commonly up- and down-regulated genes during EB differentiation and MEF reprogramming. (c-d) Top commonly up- and down-regulated genes during EB differentiation and MEF reprogramming. The y-axis shows the log2 fold change of gene expression between FLK1+ cell populations and the baseline conditions (D2.5 EB in ES/EB differentiation, and undifferentiated MEFs during MEF reprogramming). (e-f) The pathways that are significantly associated with commonly up- and down-regulated genes during ES/EB differentiation and MEF reprogramming are highlighted. The one-sided enrichment test was used to evaluate the significance of Gene Ontology enrichment. The p-value adjustment was performed by B-H Procedure.

Extended Data Fig. 6 Combined RNA-seq and ATAC-seq analysis during EB and MEF reprogramming.

(a) The number of transcription factors whose motifs associated chromatin accessibility were significantly increased or decreased in the FLK1+ cell populations at 12 hours post-Etv2 induction compared with D2.5 EBs. (b) The number of transcription factors whose motifs associated chromatin accessibility were significantly increased or decreased in the FLK1+ cell population at day 7 post-ETV2 induction compared with undifferentiated MEFs. (c-d) The number of transcription factors whose motif-associated chromatin accessibility that were commonly increased or decreased during EB and MEF reprogramming. (e) The transcription factors whose RNA-seq expression levels and motifs associated chromatin accessibility that were both up-regulated or down-regulated during EB reprogramming (FLK1+ cell from EBs at 12 hours post-ETV2 induction vs. day 2.5 EB). (f) The transcription factors whose RNA-seq expression levels and motifs associated chromatin accessibility that were both up-regulated or down-regulated during MEF reprogramming (FLK1+ cell population at day 7 post-ETV2 induction vs. undifferentiated MEFs). (g-h) The commonly up- and down-regulated genes between EBs and MEFs. (i) The transcription factor motifs that are significantly enriched in 5k region surrounding the transcription start sites of the commonly up- and down-regulated genes in EBs and MEFs. The TF enrichment was evaluated by the two-sided χ2 test in chromVAR. Data shown in 6i represent the average of two biological replicates. The p-value adjustment was performed by B-H Procedure.

Extended Data Fig. 7 The ETV2 bound sites at day 1 post-Etv2 induction in MEFs target the nucleosomes and the analysis of ETV2 ChIP-seq peaks during EB and MEF reprogramming.

(a) The MNase-seq, BRG1, H3K27ac, H3, H3K9me3, H3K27me3, H3K9ac, H3K4me3, H4K7me1 and Hdac1 ChIP-seq signals surrounding the ETV2 bound sites at day 1 post-ETV2 induction during MEF reprogramming. The ETV2 binding sites were split into nucleosome and nucleosome free region (NFR) according to the MNase-seq signals in undifferentiated MEFs. Data shown here represent the average of two biological replicates. (b) The latent representation of ATAC-seq V-plots (-320bp to + 320 bp) where the centers are nucleosome free or occupied by mono nucleosome. (c) The aggregated ETV2 bound sites centric V-plot whose centers were occupied by mono nucleosomes or nucleosome free. (d) The fragment size profile of ETV2 bound sites centric region (-320bp to +320 bp) where the centers are nucleosome free or occupied by mono nucleosomes. (e) Motif analysis of ETV2 bound sites during EB reprogramming (3 hours post-ETV2 induction) and MEF reprogramming (24 hours post-ETV2 induction). The table shows the significantly enriched motifs in ETV2 bound sites during EB and MEF reprogramming. (f) The overlap of ETV2 bound sites at day 1, day 2 and day 7 post-ETV2 induction during MEF reprogramming. (g) The overlap of ETV2 bound sites at 3 hours and 12 hours post-ETV2 induction during EB reprogramming. (h) The bar plot shows the percent of genes located near the late, early and sustained ETV2 bound sites related to blood vessel development.

Extended Data Fig. 8 Brg1 knockdown in iHA-Etv2 MEFs using shRNA lentiviral particles.

(a) Schematic diagram of shRNA lentiviral knockdown of Brg1 in iHA-Etv2 MEFs. Briefly, MEFs were exposed to shRNA particles 72 hrs before reprogramming was started and cells were collected for analysis and sequencing at various time points throughout the reprogramming process (D1, D2 and D7). shRNA particles were added throughout the reprogramming process to ensure BRG1 expression was not increased. (b) Western blot analysis of BRG1 expression in iHA-Etv2 MEFs exposed to normal reprogramming media versus media with shRNA particles against Brg1 [representative blots (b) from 3 independent experiments with similar results]. (c-l) Compared to control, shRNA knockdown of (d) Brg1 (****P < 1×10-4) in the context of (c) Etv2 overexpression (****P < 1×10-4) leads to a significant decrease in the expression of (e) Flk1 (****P < 1×10-4, ***P = 0.0005), (f) Hopx (***P = 0.0004, **P = 0.0052), (g) Otor (***P = 0.0001, ***P = 0.0009), (h) Emcn (****P < 1×10-4), (i) Sox18 (****P < 1×10-4, ***P = 0.0003), (j) Lmo2 (****P < 1×10-4), (k) Mmp9 (****P < 1×10-4, ***P = 0.001) and (l) Lax1 (****P < 1×10-4, ***P = 0.0003) transcripts, which are upregulated at D7 following overexpression of ETV2 in iHA-Etv2 MEFs (n = 3 biological replicates; one-way ANOVA with multiple comparison). Data are presented as mean ± SEM.

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Extended Data Fig. 9 Dek, Znhit1 and Cdh8 knockdown does not impact ETV2 mediated endothelial reprogramming.

(a-c) The expression profiles of (a) Dek, (b) Znhit1 and (c) Cdh8 expression during MEF reprogramming. (d-e) iHA-Etv2 MEFs were exposed to Chd8, Dek and Znhit shRNA particles 72 hrs before ETV2 mediated reprogramming was started and cells were collected for analysis 24 hrs (D1) following ETV2 overexpression. Flow cytometry analysis shows that knockdown of Chd8, Dek and Znhit does not affect MEF reprogramming mediated by ETV2 (n = 3 biological replicates). (f-h) qPCR analysis shows efficient knockdown of Chd8 (**P = 0.0068), Dek (**P = 0.0037) and Znhit (**P = 0.0017) in iHA-Etv2 MEFs (n = 3 biological replicates; one-tailed unpaired t test ****P < 1.0×10-4). Data are presented as mean ± SEM.

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Extended Data Fig. 10 ETV2 requires BRG1 to activate downstream genes during reprogramming.

(a) The heatmap shows the ATAC-seq signal surrounding the summit of 12,170 sustained ETV2 ChIP-seq peaks that were present at day 1 and day 7 post-induction of ETV2 in control MEFs (the sustained Etv2 peaks). The ATAC-seq data include undifferentiated MEFs, day 7 post-ETV2 induction, and FLK1+ cells from day 7 post-ETV2 induction in MEFs. We also include the ATAC-seq data from undifferentiated Brg1 KD (knockdown) in MEFs and day 7 post-ETV2 induction with Brg1 KD in MEFs. The sustained ETV2 peaks were divided into two groups: open (red) or closed (black) at day 7 post- induction of Brg1 KD in MEFs. (b) Heatmap shows ETV2 ChIP-seq signal surrounding 4,965 sustained ETV2 ChIP-seq peaks present in the D7 post ETV2 induction in WT MEFs and Brg1 KD MEFs. (c) UCSC genome browser tracks show the ETV2 ChIP-seq signal surrounding Etv2 ChIP-seq peaks at the promoter region of two endothelial genes Rhoj and Kdr. Data shown in 10a and 10b represent the average of two biological replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6.

Reporting Summary

Supplementary Tables 1–5

Supplementary Table 1 The transcription factors whose motif-associated chromatin accessibility and expression were upregulated in both EBs and MEFs following ETV2 induction. Supplementary Table 2 The transcription factors whose motif-associated chromatin accessibility and expression were downregulated in both EBs and MEFs following ETV2 induction. Supplementary Table 3. Taqman qPCR probes. Supplementary Table 4 Re-ChIP primers. Supplementary Table 5 ChIP qPCR primers for ELK3

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Source Data Fig. 3

Unprocessed western blot membranes. (a) Unprocessed GST blot membrane for Fig. 3d. (b) Unprocessed BRG1 blot membrane for Fig. 3e. (c) Unprocessed BRG1 blot membrane for Fig. 3f.

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Unprocessed western blot membranes. (a) Unprocessed BRG1 blot membrane for Fig. 7b. (b) Unprocessed GAPDH blot membrane for Fig. 7b.

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Unprocessed western blot membranes. (a) Unprocessed HA blot membrane for Extended Data Fig. 1c. (b) Unprocessed GAPDH blot membrane for Extended Data Fig. 1c.

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Unprocessed western blot membranes. (a) Unprocessed BRG1 blot membrane for Extended Data Fig. 8b. (b) Unprocessed GAPDH blot membrane for Extended Data Fig. 8b.

Source Data Extended Data Fig. 9

Statistical source data.

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Gong, W., Das, S., Sierra-Pagan, J.E. et al. ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage. Nat Cell Biol 24, 672–684 (2022). https://doi.org/10.1038/s41556-022-00901-3

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