Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
- University of California, San Diego, United States
- University of Arizona, United States
- University of Western Michigan, United States
Figures
Figure 1 with 3 supplements
HAECs display a distinct repertoire of enhancers that nominate combinations of the AP1, ETS, SOX and GATA TF families as major orchestrators of HAEC gene expression.
(a) A heatmap of 16,929 enhancer-like regions were selected by: accessible chromatin (ATAC-seq), coincident with H3K4me2 and H3K27ac deposition (ChIP-seq) in gene-distal positions (≥3 kb from …
Figure 1—figure supplement 1
Analysis of motifs and expression of associated transcription factors.
(a) The Top 19 enriched motifs that occur in active endothelial cell enhancers (Figure 1a) calculated from 200 bp sequences centered on chromatin accessible regions. (b) Ranked TF gene expression …
Figure 1—figure supplement 2
Genetic loci for JUN, JUNB, and JUND.
Chromatin accessibility, histone modifications, and RNA expression of JUN, JUNB, and JUND. SE regions are highlighted in yellow as determined by H3K27ac.
Figure 1—figure supplement 3
Hierarchical clustering of enhancers.
(a) H3K27ac ChIP-seq datasets were downloaded from ENCODE and Roadmap Epigenomics and quantified at 16,929 HAEC enhancers from Figure 1a (x-axis). Enhancers are clustered using hierarchical …
Figure 2 with 2 supplements
Coordinate gene expression across 96 genetically distinct HAEC donors identifies three regulatory programs among ETS, AP1, SOX, and GATA family members.
(a) TF gene expression measured by AffyHU133A array is shown across a population of 97 unique HAEC donors. Array probe set IDs were manually confirmed to cover expressed transcript isoforms of the …
Figure 2—figure supplement 1
The HAEC gene correlation network.
A clustered heatmap of correlation coefficients calculated between the TFs of interest (x-axis) and 7738 transcripts having significant correlation to at least one TF (Pearson correlation p<0.05) on …
Figure 2—figure supplement 2
TF binding at HAEC enhancers.
JUNB, JUN, GATA2, ERG, and ETS1 bind at the center of the enhancer-like regions (from Figure 1a). JUNB, JUN, and ERG binding was measured in HAECs and GATA2 and ETS1 in HUVECs.
Figure 3 with 2 supplements
ERG and JUN co-bind EC enhancers and are enriched at EC-specific genes.
(a–c) Promoter-distal regions bound by ERG, JUN, and JUNB, or GATA2 are shown in a, b, and c respectively in a one kilobase window. Each set was centered on the corresponding binding motif, and the …
Figure 3—figure supplement 1
Allele-specific JUN binding at loci with mutated motifs.
JUN binding was measured by ChIP-seq in a HAEC donor with whole-genome sequencing information. Allele-specific JUN binding was calculated at heterozygous loci (y-axis is JUN binding ratio between …
Figure 3—figure supplement 2
JUN and ERG co-occupy loci near EC-specific genes.
ATAC-seq, binding for JUN and ERG, histone modifications H3K4me2 and H3K27ac, and RNA abundance are shown at the genetic loci for CDH5, EGFL7, TEK/TIE2, and PECAM1. Promoters are highlighted in …
Figure 4 with 2 supplements
ERG knockdown elicits a pro-inflammatory gene profile in HAECs.
(a) Genes up-regulated by ERG knockdown (≥4 fold, 5% FDR) were enriched in the indicated functional pathways using Ingenuity Pathway Analysis (IPA). Results are from three experiments using cells …
Figure 4—figure supplement 1
Changes in gene expression with ERG knockdown measured by RNA-seq.
Gene expression levels of various genes according to RNA-seq data in three different donors, each in control (siSCR) and ERG knockdown (siERG) conditions.
Figure 4—figure supplement 2
Changes in gene expression with ERG knockdown measured by RT qPCR.
Gene expression measured by RT- qPCR in no transfection controls, with scrambled siRNA, individual siERG oligos, and pooled siERG oligos for genes that either inflammatory or endothelial-specific …
Figure 5 with 4 supplements
Inflammatory signals activate enhancer-like elements with distinct motifs and suggest that CEBP and IRF members mediate responsiveness to TNFα and IL1β.
(a) A schematic for de novo enhancer activation by combinations of signal-dependent and collaborative TFs. (b) Enrichments of the NRF2, NFκB, IRF, C/EBP, AP1, and ETS motifs (y-axis) are shown for …
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Figure 5—source data 1
Motif enrichments in 100 bp sequences defined by promoter-distal (≥3 kb) loci gaining ATAC-seq and H3K27ac upon 4 hr oxPAPC, TNFα, and IL1β treatments.
- https://doi.org/10.7554/eLife.22536.018
Figure 5—figure supplement 1
The transcriptional response to oxPAPC, TNFα, and IL1β.
(a) Enriched pathways for genes regulated after 4-hr treatment with 40 µg/ml oxPAPC, 10 ng/ml IL1β, and 2 ng/ml TNFα are shown. (b) The proportion of molecule types regulated per cell treatment. (c–g…
Figure 5—figure supplement 2
Signal-responsive transcription factor NRF2 binds endothelial enhancers.
(a) HMOX1, TXNRD1, NQO1, and GCLM loci are co-bound by ERG, JUN and NRF2 and acquire H3K27ac upon oxPAPC treatment. (b) NRF2 binding was measured by ChIP-seq after oxPAPC treatment and coincides …
Figure 5—figure supplement 3
Signal-responsive transcription factor NFκB binds endothelial enhancers.
(a) The heme oxygenase 1 (HMOX1) and thioredoxin reductase 1 (TXNRD1) loci are co-bound by ERG, JUN and NRF2 and accumulate H3K27ac upon 4-hr oxPAPC treatment. (b) NRF2 binding was measured by …
Figure 5—figure supplement 4
Binding of p65 at mutated motifs at each allele p65 (NFκB) binding was measured by ChIP-seq in a HAEC donor with whole-genome sequencing information.
Allele-specific p65 binding was calculated at heterozygous loci (y-axis is p65 binding ratio between alleles). Allele-specific motif mutations were calculated as instances where an allele mutated …
Figure 6 with 1 supplement
CEBPD and IRF1 knockdown effect on gene expression response to TNFα.
(a) Binding of p65, CEBPD, and IRF1 was measured by ChIP-seq and is shown at de novo enhancers elicited by IL1β (e) and TNFα (f). Factor binding was measured by CHIP-seq upon 4-hr cytokine …
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Figure 6—source data 1
Transcripts up-regulated by more than twofold by CEBPD knockdown in untreated HAECs compared to scrambled control.
Differential expression performed in DEseq.
- https://doi.org/10.7554/eLife.22536.024
Figure 6—figure supplement 1
ERG and p65 co-bind loci with a distinctive ETS/NFκB motif.
(a) ERG mRNA was measured in HAECs by RNA-seq in three independent experiments in media lacking (no treatment) or containing 2 ng/µl TNFα or 10 ng/µl IL1β for 4 hr. (b) ATAC-seq, RNA-seq, and …
Tables
Table 1
Overlap of HAEC enhancers with GWAS loci reported for coronary artery disease (CAD) or hypertension (HT). Associated SNPs were downloaded from the NHGRI-EBI Catalog of published genome-wide …
| GWAS SNP | HAEC enhancer | |||||||
|---|---|---|---|---|---|---|---|---|
| SNP in enhancer | LD to lead SNP from study | p-Value of lead | Trait | Reported gene of lead | PubMed ID | Position (chr, start bp, end bp) | Nearest gene | Type |
| rs12091564 | Lead | 2.0E-07 | CAD | HFE2 | 21626137 | 1, 145395579, 145395699 | LOC101928979 | common |
| rs72701850 | LD, rs12091564, r2 = 0.95346 | 2.0E-07 | CAD | HFE2 | 21626137 | 1, 145396840, 145397006 | LOC101928979 | common |
| rs72701850 | LD, rs10218795, r2 = 0.95346 | 2.0E-07 | CAD | HFE2 | 21626137 | 1, 145396840, 145397006 | LOC101928979 | common |
| rs56348932 | LD, rs17114036, r2 = 0.916823 | 4.0E-19 | CAD | PLPP3 | 21378990, 24262325 | 1, 56988477, 56988661 | PLPP3 | EC-specific |
| rs56348932 | LD, rs9970807, r2 = 0.942868 | 2.0E-09 | CAD | PLPP3 | 26343387 | 1, 56988477, 56988661 | PLPP3 | EC-specific |
| rs56348932 | LD, rs17114046, r2 = 0.942868 | 3.0E-07 | CAD | PLPP3 | 21846871, 21378988 | 1, 56988477, 56988661 | PLPP3 | EC-specific |
| rs10047079 | LD, rs2229238, r2 = 0.866848 | 7.0E-07 | CAD | ILR6 | 22319020 | 1, 154468114, 154468189 | SHE | EC-specific |
| rs55916033 | LD, rs10496288, r2 = 1 | 2.0E-09 | HT | intergenic | 21626137 | 2, 83278987, 83279062 | LOC1720 | EC-specific |
| rs55916033 | LD, rs10496289, r2 = 1 | 2.0E-09 | HT | intergenic | 21626137 | 2, 83278987, 83279062 | LOC1720 | EC-specific |
| rs72836880 | LD, rs10496288, r2 = 1 | 2.0E-09 | HT | intergenic | 21626137 | 2, 83308909, 83309314 | LOC1720 | EC-specific |
| rs72836880 | LD, rs10496289, r2 = 1 | 2.0E-09 | HT | intergenic | 21626137 | 2, 83308909, 83309314 | LOC1720 | EC-specific |
| rs112798061 | LD, rs10496289, r2 = 1 | 2.0E-09 | HT | intergenic | 21626137 | 2, 83308909, 83309314 | LOC1720 | EC-specific |
| rs3748861 | LD, rs13420028, r2 = 0.916266 | 1.0E-10 | HT | GPR39 | 21626137 | 2, 133196310, 133196505 | GPR39 | mix |
| rs3748861 | LD, rs10188442, r2 = 0.916266 | 1.0E-10 | HT | GPR39 | 21626137 | 2, 133196310, 133196505 | GPR39 | mix |
| rs144505847 | LD, rs6725887, r2 = 1 | 1.0E-09 | CAD | WDR12 | 21378990, 24262325 | 2, 203672243, 203672412 | ICA1L | EC-specific |
| rs144505847 | LD, rs7582720, r2 = 1 | 3.0E-08 | CAD | WDR12 | 24262325 | 2, 203672243, 203672412 | ICA1L | EC-specific |
| rs56155140 | LD, rs17087335, r2 = 0.979112 | 5.0E-08 | CAD | NOA1, REST | 26343387 | 4, 57824385, 57824541 | NOA1 | mix |
| rs5869162 | LD, rs6452524, r2 = 0.924698 | 2.0E-07 | HT | XRCC4 | 21626137 | 5, 82393827, 82393921 | XRCC4 | EC-specific |
| rs5869162 | LD, rs6887846, r2 = 0.924698 | 2.0E-07 | HT | XRCC4 | 21626137 | 5, 82393827, 82393921 | XRCC4 | EC-specific |
| rs6475604 | LD, rs7865618, r2 = 0.940597 | 2.0E-27 | CAD | MTAP | 21606135 | 9, 22052677, 22052823 | CDKN2B | EC-specific |
| rs17293632 | LD, rs72743461, r2 = 1 | 1.0E-07 | CAD | SMAD3 | 26343387 | 15, 67442510, 67442670 | SMAD3 | common |
| rs17293632 | LD, rs56062135, r2 = 0.988489 | 5.0E-09 | CAD | SMAD3 | 26343387 | 15, 67442510, 67442670 | SMAD3 | common |
| rs17227883 | LD, rs17228212, r2 = 0.981438 | 2.0E-07 | CAD | SMAD3 | 17634449 | 15, 67442769, 67443128 | SMAD3 | common |
| rs1563966 | LD, rs1231206, r2 = 0.844151 | 9.0E-10 | CAD | intergenic | 21378990 | 17, 2095878, 2096222 | LOC101927839 | mix |
| rs1563966 | LD, rs216172, r2 = 0.909315 | 1.0E-09 | CAD | SMG6, SRR | 21378990, 26343387 | 17, 2095878, 2096222 | LOC101927839 | mix |
| rs7408563 | LD, rs7246657, r2 = 0.900512 | 7.0E-06 | CAD | ZNF383 | 23870195 | 19, 37808501, 37809067 | HKR1 | common |
Table 2
Molecular trait changes observed upon HAEC exposure to pro-inflammatory stimuli. Differential expression was determined in DESeq with duplicate RNA-seq experiments.
| Stimulus | Regulated genes (>2 fold, 5%FDR) | de novo enhancers increased H3K27ac and accessibility upon stimulation |
|---|---|---|
| oxPAPC 4 hr (40 µg/ml) versus control | 322 (242 up, 80 down) | 839 |
| TNFα 4 hr (2 ng/ml) versus control | 840 (611 up, 229 down) | 266 |
| IL1β 4 hr (10 ng/ml) versus control | 1174 (807 up, 367 down) | 3199 |
Additional files
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Supplementary file 1
Public H3K27ac ChIP-seq datasets used to define endothelial-specific enhancer-like regions.
The GEO accession number and cell or tissue type is listed for each dataset in the analysis.
- https://doi.org/10.7554/eLife.22536.026
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Supplementary file 2
Knockdown of ERG with siRNA modulates transcription of numerous other HAEC transcription factors.
Above are the transcription factors shown to be modulated in two separate HAEC donors (by poly-A RNA-sequencing, greater than twofold change in transcript level, adjusted p<0.05).
- https://doi.org/10.7554/eLife.22536.027
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Supplementary file 3
List of primers and siRNA oligos used in study
- https://doi.org/10.7554/eLife.22536.028
