gongx030/Etv2_pioneer

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

The code and datasets for the manuscript:

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

Wuming Gong, Satyabrata Das, Javier E. Sierra-Pagan, Erik Skie, Nikita Dsouza, Thijs A. Larson, Mary G. Garry, Edgar Luzete-Monteiro, Kenneth S. Zaret, Daniel J. Garry

Processed datasets

	Dataset	Format	Script
ATAC-seq counts	link	SummarizedExperiment	R
scRNA-seq of Etv2 induced reprogramming	link	SummarizedExperiment	R
Bulk RNA-seq of EB differentiation induced by Etv2 Dox	link	SummarizedExperiment
A union set of Etv2 ChIP-seq peaks in ES/EB and MEFs	link	SummarizedExperiment	R
Preprocessing Etv2 ChIP-seq, H3K27ac ChIP-seq and Brg1 ChIP-seq in ES/EB and MEF	link		R
Bash script for preprocessing the Brg1 KO H3K27ac ChIP-seq in MEF			bash
Fastq datasets for ChIP-seq, ATAC-seq, bulkRNA-seq, scRNA-seq and NOMeseq	link

Main Figures

Figure 1

	Figures	Colab link
The UMAP plot for the scRNA-seq of 948 undifferentiated MEFs, 3,539 reprogrammed cells at 24 hrs, 2,936 cells at 48 hrs and 7,202 cells at 7 days and 827 FLK1+/KDR cells at 7 days post-induction of ETV2 in MEFs	Fig. 1c	R
The UMAP plot showing cell clusters from k-means clustering that identified seven distinct cell clusters	Fig. 1d	R
The expression profiles of ETV2 and FLK1/KDR	Fig. 1e	R
The volcano plot of genes differentially expressed between cluster 1 and cluster 7	Fig. 1f	R
The PCA of the variations of transcription factor (TF) associated chromatin accessibility of the ATAC-seq of MEF reprogramming (MEFs, 24 hrs, 48 hrs and 7 days post-induction) and EB differentiation (2.5 days and 3 hrs post induction)	Fig. 1h	R
The 31 TF expression levels and motif associated chromatin accessibility consistently showed directional change in both EBs and MEFs (13 up-regulated TFs and 18 down-regulated TFs)	Fig. 1i	R

Figure 2

	Figures	Colab link
The Venn diagram shows 131,001 and 18,024 Etv2 ChIP-seq peaks at 24 hrs post-induction during MEF reprogramming and 3 hrs post-induction in day 2.5 EBs, respectively	Fig. 2a	R
The genomic distribution of EB specific, MEF specific and common ETV2 peaks. The EB and MEF specific ETV2 peaks were more likely distributed at the distal intergenic regions	Fig. 2b	R
The heatmap shows the read density of MNase-seq, BRG1 ChIP-seq and H3K27ac ChIP-seq in MEFs, surrounding 131,001 ETV2 ChIP-seq peaks at 24 hrs post-induction during MEF reprogramming. The ETV2 peaks were divided into four quartiles based on the mean MNase-seq signals of the central 200-bp region	Fig. 2c	R
The heatmap shows the ratio of NFR / nucleosome read density, read density of BRG1 ChIP-seq and H3K27ac ChIP-seq in EBs (day 2.5), surrounding 18,024 ETV2 ChIP-seq at 3 hrs post-induction. The ETV2 peaks were divided into NFR (5,291 peaks) and nucleosome (8,843 peaks) groups according to the local V-plot and fragment size profiles of ATAC-seq day 2.5 EBs without ETV2 induction	Fig. 2d	R
The heatmap generated using NOMe-seq shows among 5,320 ETV2 binding sites that were nucleosome occupied at undifferentiated MEF, 4,744 (89.1%) became significantly nucleosome-free (NFR) while 576 (10.9%) stayed NOR at D1 of reprogramming	Fig. 2e	R
Sequence motif analysis by DREME and CentriMo identified a common GGAAAT motif that were significantly more enriched in NFR regions compared with the nucleosomes in both MEFs and EBs	Fig. 2f	R
A region upstream of Lmo2 that was highly enriched for nucleosomes in both cell types, as measured by MNase-seq and ATAC-seq was selected to perform in vitro nucleosomal binding assays	Fig. 2g	R

Figure 3

	Figures	Colab link
The average partial Etv2 motif scores in upstream/downstream 500 bp regions surrounding the binding summit of the "early', "late" and "sustained" Etv2 peaks in EB differentiation and MEF reprogramming	Fig. 3a	R
The percent of the "early', "late" and "sustained" Etv2 peaks in EBs and MEFs include partial Etv2 motifs in upstream/downstream 50bp regions surrounding the binding summits	Fig. 3b	R
The heatmap shows the fold enrichment of ETV2 ChIP-seq, BRG1 ChIP-seq and H3K27ac ChIP-seq at 24 hrs, 48 hrs and 7 days post-induction of ETV2	Fig. 3g	R
The heatmap shows the fold enrichment of ETV2 ChIP-seq, BRG1 ChIP-seq and H3K27ac ChIP-seq, at 3 hrs and 12 hrs post-induction of ETV2 in day 2 EBs	Fig. 3h	R
The Venn diagram shows the overlap between late ETV2 peaks in MEFs and EBs, as well as the overlap between the genes near the late ETV2 peaks	Fig. 3j	R

Figure 4

	Figures	Colab link
VAE to 18,214 Etv2 ChIP-seq peaks during MEF reprogramming and identified six clusters of V-plot according to the central fragment size distribution	Fig. 4a	R
The six clusters included three types of V-plots where the central Etv2 sites were nucleosome free (C1, C3 and C4), and three types of V-plots where the central Etv2 sites were nucleosome occupied (C2, C5, and C6), represented by aggregated V-plot	Fig. 4b	R
The six clusters included three types of V-plots where the central Etv2 sites were nucleosome free (C1, C3 and C4), and three types of V-plots where the central Etv2 sites were nucleosome occupied (C2, C5, and C6), represented by NucleoATAC	Fig. 4c	R
The six clusters included three types of V-plots where the central Etv2 sites were nucleosome free (C1, C3 and C4), and three types of V-plots where the central Etv2 sites were nucleosome occupied (C2, C5, and C6), represented by aggregated V-plot	Fig. 4d	R
The six clusters included three types of V-plots where the central Etv2 sites were nucleosome free (C1, C3 and C4), and three types of V-plots where the central Etv2 sites were nucleosome occupied (C2, C5, and C6), represented by NucleoATAC	Fig. 4e	R
The bar plot shows the proportion of each V-plot clusters in early, late and sustained Etv2 peaks, as well as in the background peaks	Fig. 4f	R
The cluster labels of early, late and sustained ETV2 peak that were changed from MEF to D7 Flk1+ cells	Fig. 4g	R
The fragment size distribution of ATAC-seq of FLK1+ cells vs. the mixture population at 12 hours post-ETV2 induction during EB reprogramming and day 7 post-ETV2 induction during MEF reprogramming. In both conditions, the mono-nucleosomes and the di-nucleosomes were significantly increased in the FLK1+ cell populations	Fig. 4h	R
The aggregated V-plot whose centers are the ETV2 bound sites at FLK1+ cell populations at 12 hours post-ETV2 induction in EB, and at 7 days post-ETV2 induction	Fig. 4i	R

Figure 5

	Figures	Colab link
The UMAP plot shows the scRNA-seq of 8,838 cells from day 7 post-induction in control MEFs, 1,502 FLK1+ cells from day 7 post-ETV2 induction in MEFs, 8,248 cells from day 7 post-ETV2 induction in Brg1 KD MEFs, and 8,034 cells at day 7 in Brg1 KD MEFs	Fig. 5c	R
The UMAP plot shows the expression level of Etv2	Fig. 5d	R
The UMAP plot shows the expression level of Brg1	Fig. 5e	R
The UMAP plot shows the expression level of Flk1	Fig. 5f	R

Figure 6

	Figures	Colab link
The heatmap shows the piled up 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)	Fig. 6a	R
The heatmap shows the transcription factors where motif associated chromatin accessibility were significantly changed at day 7 post-ETV2 induction in MEFs (unsorted MEFs or FLK1+ cells), or the Brg1 KD MEFs	Fig. 6b	R
The genomic distribution of open ETV2 peaks and closed ETV2 peaks at 7 days post-ETV2 induction in Brg1 KD MEFs	Fig. 6c	R
The aggregated V-plot of 1,204 ETV2 binding sites that were closed in MEFs and became open at day 1 post-ETV2 induction in MEFs	Fig. 6d	R

Figure 7

	Figures	Colab link
The heatmap shows chromatin accessibility for transcription factors for control vs. Brg1 knockout during ES/EB differentiation	Fig. 7f	R
The heatmap shows the ChIP-seq of sustained ETV2 binding sites in EB differentiation, and ATAC-seq of Control and Brg1 KO EBs at day 4	Fig. 7g	R
ATAC-seq V-plots of the genomic regions (640 bp) that are centered at ELK3 motifs	Fig. 7h	R

Extended Figures

Extended Figure 1

	Figures	Colab link
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)	Extended Data Fig. 1h	R

Extended Figure 2

	Figures	Colab link
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	Extended Data Fig. 2a	R
The violin plots show the scaled expression levels of endothelial markers in seven cell clusters	Extended Data Fig. 2b	R
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	Extended Data Fig. 2c	R
GSEA plot indicates significant upregulation of the inflammatory response in MEFs (cluster 1)	Extended Data Fig. 2d	R
Heatmap representing the gene expression levels scaled by Seurat for upregulated (red) and downregulated (blue) genes in cluster 1 and cluster 2	Extended Data Fig. 2e	R
The bar plots show top 10 significant pathways for cluster 1 MEFs	Extended Data Fig. 2f	R
The bar plots show top 10 significant pathways for cluster 2 MEFs.	Extended Data Fig. 2g	R

Extended Figure 3

	Figures	Colab link
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, day2, day 7 and Flk1+ cells at day 7	Extended Data Fig. 3a	R
The bar plot shows immune response related pathways significantly upregulated in Flk1+ cells from day 7 post Etv2 induction compared to MEFs	Extended Data Fig. 3b	R
The bar plot shows immune response related pathways significantly upregulated in ay 1 post Etv2 induction compared to MEFs	Extended Data Fig. 3c	R

Extended Figure 5

	Figures	Colab link
The Venn diagrams show the overlap of commonly up-regulated genes during EB differentiation and MEF reprogramming	Extended Data Fig. 5a	R
The Venn diagrams show the overlap of commonly down-regulated genes during EB differentiation and MEF reprogramming	Extended Data Fig. 5b	R
Top commonly up-regulated genes during EB differentiation and MEF reprogramming	Extended Data Fig. 5c	R
Top commonly down-regulated genes during EB differentiation and MEF reprogramming	Extended Data Fig. 5d	R
The pathways that are significantly associated with commonly up-regulated genes during ES/EB differentiation and MEF reprogramming	Extended Data Fig. 5e	R
The pathways that are significantly associated with commonly down-regulated genes during ES/EB differentiation and MEF reprogramming	Extended Data Fig. 5f	R

Extended Figure 6

	Figures	Colab link
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	Extended Data Fig. 6a	R
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	Extended Data Fig. 6b	R
The number of transcription factors whose motif associated chromatin accessibility that were commonly increased during EB and MEF reprogramming	Extended Data Fig. 6c	R
The number of transcription factors whose motif associated chromatin accessibility that were commonly decreased during EB and MEF reprogramming	Extended Data Fig. 6d	R
The transcription factors whose RNA-seq expression levels and motifs associated chromatin accessibility that were both up-regulated or down-regulated during EB reprogramming	Extended Data Fig. 6e	R
The transcription factors whose RNA-seq expression levels and motifs associated chromatin accessibility that were both up-regulated or down-regulated during MEF reprogramming	Extended Data Fig. 6f	R
The commonly up-regulated genes between EBs and MEFs	Extended Data Fig. 6g	R
The commonly down-regulated genes between EBs and MEFs	Extended Data Fig. 6h	R
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	Extended Data Fig. 6i	R

Extended Figure 7

	Figures	Colab link
The MNase-seq, BRG1, H3K27ac, H3, H3K9me3, H3K27me3, H3K9ac, H3K4me3, H4K7me1 and Hdac1 ChIP-seq signals surrounding the ETV2 bound sites at day1 post-ETV2 induction during MEF reprogramming, split into nucleosome and nucleosome free region (NFR) according to the MNase-seq signals	Extended Data Fig. 7a	R
The latent representation of ATAC-seq V-plots (-320bp to + 320bp) where the centers are nucleosome free or occupied by mono nucleosome	Extended Data Fig. 7b	R
The aggregated ETV2 bound sites centric V-plot whose centers were occupied by mono nucleosomes or nucleosome free	Extended Data Fig. 7c	R
The fragment size profile of ETV2 bound sites centric region (-320bp to +320bp) where the centers are nucleosome free or occupied by mono nucleosomes	Extended Data Fig. 7d	R
Motif analysis of ETV2 bound sites during EB reprogramming and MEF reprogramming	Extended Data Fig. 7e	R
The overlap of ETV2 bound sites at day 1, day 2 and day 7 post-ETV2 induction during MEF reprogramming	Extended Data Fig. 7f	R
The overlap of ETV2 bound sites at 3 hours and 12 hours post-ETV2 induction during EB reprogramming	Extended Data Fig. 7g	R
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. 7h	R

Extended Figure 9

	Figures	Colab link
The expression profile of Dek expression during MEF reprogramming	Extended Data Fig. 9a	R
The expression profile of Znhit1 expression during MEF reprogramming	Extended Data Fig. 9b	R
The expression profile of Chd8 expression during MEF reprogramming	Extended Data Fig. 9c	R

Extended Figure 10

	Figures	Colab link
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	Extended Data Fig. 10a	R
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	Extended Data Fig. 10b	R

Supplementary Figure

Supplementary Figure 1

	Figures	Colab link
The plots show enrichment scores for each Histone Acetyltransferases'	Supplementary Fig. 1a	R
The plots show enrichment scores for each Histone Deacetylases'	Supplementary Fig. 1b	R
The plots show enrichment scores for each Inflammatory Response	Supplementary Fig. 1c	R
The plots show enrichment scores for each NIK/NF-kappaB signaling	Supplementary Fig. 1d	R

Supplementary Figure 2

	Figures	Colab link
The plots show enrichment scores for each Histone Acetyltransferases'	Supplementary Fig. 2a	R R
The plots show enrichment scores for each Histone Deacetylases'	Supplementary Fig. 2b	R R
The plots show enrichment scores for each Inflammatory Response	Supplementary Fig. 2c	R R
The plots show enrichment scores for each NIK/NF-kappaB signaling	Supplementary Fig. 2d	R R

Supplementary Figure 3

	Figures	Colab link
The plots show enrichment scores for each Histone Acetyltransferases'	Supplementary Fig. 3a	R
The plots show enrichment scores for each Histone Deacetylases'	Supplementary Fig. 3b	R
The plots show enrichment scores for each Inflammatory Response	Supplementary Fig. 3c	R
The plots show enrichment scores for each NIK/NF-kappaB signaling	Supplementary Fig. 3d	R

Supplementary Figure 4

	Figures	Colab link
Venn diagram representing the overlap of common genes upregulated in ES/EB and MEF reprogramming post Etv2 induction, also overlapping with the genes from gene ontology terms for Histone Acetyltransferases'	Supplementary Fig. 4a	R
Venn diagram representing the overlap of common genes upregulated in ES/EB and MEF reprogramming post Etv2 induction, also overlapping with the genes from gene ontology terms for Histone Deacetylases'	Supplementary Fig. 4b	R
Venn diagram representing the overlap of common genes upregulated in ES/EB and MEF reprogramming post Etv2 induction, also overlapping with the genes from gene ontology terms for Inflammatory Response	Supplementary Fig. 4c	R
Venn diagram representing the overlap of common genes upregulated in ES/EB and MEF reprogramming post Etv2 induction, also overlapping with the genes from gene ontology terms for NIK/NF-kappaB signaling	Supplementary Fig. 4d	R

Supplementary Figure 5

	Figures	Colab link
The transcription factors whose motif associated chromatin accessibility and expressions were up-regulated in both EB and MEF on Etv2 induction	Supplementary Fig. 5a	R
The transcription factors whose motif associated chromatin accessibility and expressions were down-regulated in both EB and MEF on Etv2 induction	Supplementary Fig. 5b	R

Supplementary Figure 6

	Figures	Colab link
The PCA analysis of undifferentiated MEFs and Brg1 KD MEFs	Supplementary Fig. 6a	R
The PCA analysis of different cell cycle programs	Supplementary Fig. 6b	R
The bar plot showing cells in the G1 phase are increased with Brg1 KD in MEFs	Supplementary Fig. 6c	R
The correction of cell cycle effects when combining the single cell RNA-seq data of undifferentiated MEFs and Brg1 KD MEFs	Supplementary Fig. 6d	R
The correction of cell cycle effects when combining the single cell RNA-seq data of undifferentiated MEFs and Brg1 KD MEFs	Supplementary Fig. 6e	R
The expression levels of Etv2 in cell-cycle effected adjusted single cell RNA-seq data from undifferentiated MEFs and Brg1 KD MEFs	Supplementary Fig. 6f	R
The expression levels of Brg1 in cell-cycle effected adjusted single cell RNA-seq data from undifferentiated MEFs and Brg1 KD MEFs	Supplementary Fig. 6g	R
The H3K27ac ChIP-seq signals of undifferentiated MEFs and Brg1 KD MEFs surrounding the early, late and sustained ETV2 bound sites in ETV2 induced MEF reprogramming	Supplementary Fig. 6h	R

GEO datasets

The datasets below belong to a SuperSeries GSE185684

Assay Type	GSE
ATACseq	GSE168636
ChIPseq	GSE168521
RNA-seq	GSE185682
scRNA-seq	GSE185683
NOMe-seq	GSE185681

Mass Spectrometry data

The Mass Spectrometry data of ETV2 interacting proteins in D4 EBs.

https://github.com/gongx030/Etv2_pioneer/blob/master/massspec.xlsx

UCSC genome browser track

	Figures	UCSC Genome browser
Lmo2 locus	Figure 2g	Track
All ChIP-seq data		Track
Rhoj promoter	Figure 14c	Track
Kdr promoter	Figure 14c	Track