An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability
- University of Cambridge, United Kingdom
- University of Oxford, United Kingdom
- NHS Blood and Transplant, United Kingdom
- VU University Amsterdam, Netherlands
- Netherlands Cancer Institute, Netherlands
Figures
Figure 1 with 8 supplements
Identification of haematopoietic active cis-regulatory regions.
(a) UCSC screenshot of the Erg gene locus for ChIP-Sequencing data for nine haematopoietic TFs (ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 [Wilson et al., 2010]) and for H3K27ac (Cale…
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Figure 1—source data 1
Number of PCR and LacZ positive transgenic embryos (E10.5–11.5) for each regulatory region.
- https://doi.org/10.7554/eLife.11469.004
Figure 1—figure supplement 1
Identification of haematopoietic active cis-regulatory elements for Fli1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 2
Identification of haematopoietic active cis-regulatory elements for Gata2.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 3
Identification of haematopoietic active cis-regulatory elements for Gfi1b.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 4
Identification of haematopoietic active cis-regulatory elements for Lyl1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 5
Identification of haematopoietic active cis-regulatory elements for Meis1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 6
Identification of haematopoietic active cis-regulatory elements for Runx1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 7
Identification of haematopoietic active cis-regulatory elements for Spi1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 1—figure supplement 8
Identification of haematopoietic active cis-regulatory elements for Tal1.
(a) The candidate cis-regulatory elements were identified by ChIP-Seq analysis of the TFs ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1 as well as H3K27 acetylation in the …
Figure 2 with 8 supplements
Comparison of TF binding pattern at haematopoietic active cis-regulatory regions in two haematopoietic progenitor cell lines, HPC7 and 416b.
(a) UCSC screenshot of the Erg gene locus for ChIP-Sequencing data for nine haematopoietic TFs (ERG, FLI1, GATA2, GFI1B, LYL1, MEIS1, PU.1, RUNX1 and TAL1) and for H3K27ac in 416b cells. Highlighted …
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Figure 2—source data 1
List of ChIP-Seq samples included in the heatmap in Figure 2b.
- https://doi.org/10.7554/eLife.11469.014
Figure 2—figure supplement 1
UCSC screenshot for the Fli1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink are cis-regulatory regions that were identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and were shown to possess haematopoietic activity. The …
Figure 2—figure supplement 2
UCSC screenshot for the Gata2 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink are cis-regulatory regions that were identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and were shown to possess haematopoietic activity. The …
Figure 2—figure supplement 3
UCSC screenshot for the Gfi1b gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink are cis-regulatory regions that were identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and were shown to possess haematopoietic activity. The …
Figure 2—figure supplement 4
UCSC screenshot for the Lyl1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink is the promoter ('pro') that was identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and was shown to possess haematopoietic activity. The …
Figure 2—figure supplement 5
UCSC screenshot for the Meis1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink is the cis-regulatory region that was identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and was shown to possess haematopoietic activity. The …
Figure 2—figure supplement 6
UCSC screenshot for the Runx1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink are cis-regulatory regions that were identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and were subsequently shown to possess haematopoietic …
Figure 2—figure supplement 7
UCSC screenshot for the Spi1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink is the cis-regulatory region that was identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and was shown to possess haematopoietic activity. The …
Figure 2—figure supplement 8
UCSC screenshot for the Tal1 gene locus demonstrating binding patterns for nine key haematopoietic TFs and H3K27ac in 416b cells.
Highlighted in pink are cis-regulatory regions that were identified based on the selection criteria (≥3 TFs bound and H3K27ac) in HPC7 cells and were shown to possess haematopoietic activity. The …
Figure 3 with 18 supplements
TFBS mutagenesis reveals enhancer-dependent effects of TF binding on gene expression.
(a) Multiple species alignment of mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1) sequences for the Erg+65 region. Nucleotides highlighted in black are conserved …
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Figure 3—source data 1
List of TF binding sites and the TFs that bind to them.
- https://doi.org/10.7554/eLife.11469.024
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Figure 3—source data 2
List of co-ordinates and primer sequences for the regulatory regions analysed in this study.
- https://doi.org/10.7554/eLife.11469.025
Figure 3—figure supplement 1
Multiple species alignment and luciferase assay results for Erg+75.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 2
Multiple species alignment and luciferase assay results for Erg+85.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 3
Multiple species alignment and luciferase assay results for Fli1+12.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 4
Multiple species alignment and luciferase assay results for Gata2-93.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 5
Multiple species alignment and luciferase assay results for Gata2+3.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 6
Multiple species alignment and luciferase assay results for Gfi1b+16.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 7
Multiple species alignment and luciferase assay results for Gfi1b+17.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 8
Multiple species alignment and luciferase assay results for Lyl1 promoter.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2) and opossum (monDom5). Nucleotides highlighted in black are conserved between all species …
Figure 3—figure supplement 9
Multiple species alignment and luciferase assay results for Meis1+48.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 10
Multiple species alignment and luciferase assay results for Spi1-14.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 11
Multiple species alignment and luciferase assay results for Runx1-59.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19) and dog (canFam2). Nucleotides highlighted in black are conserved between all species analysed, nucleotides …
Figure 3—figure supplement 12
Multiple species alignment and luciferase assay results for Runx1+3.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 13
Multiple species alignment and luciferase assay results for Runx1+23.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2) and opossum (monDom5). Nucleotides highlighted in black are conserved between all species …
Figure 3—figure supplement 14
Multiple species alignment and luciferase assay results for Runx1+110.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 15
Multiple species alignment and luciferase assay results for Runx1+204.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2), opossum (monDom5) and platypus (ornAna1). Nucleotides highlighted in black are conserved …
Figure 3—figure supplement 16
Multiple species alignment and luciferase assay results for Tal1-4.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19) and dog (canFam2). Nucleotides highlighted in black are conserved between all species analysed, nucleotides …
Figure 3—figure supplement 17
Multiple species alignment and luciferase assay results for Tal1+19.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19), dog (canFam2) and opossum (monDom5). Nucleotides highlighted in black are conserved between all species …
Figure 3—figure supplement 18
Multiple species alignment and luciferase assay results for Tal1+40.
(a) Multiple species alignment (MSA) with the following species: mouse (mm9), human (hg19) and dog (canFam2). Nucleotides highlighted in black are conserved between all species analysed, nucleotides …
Figure 4 with 1 supplement
A three-tier dynamic Bayesian network (DBN) incorporating transcriptional regulatory information can recapitulate the HSPC expression state.
(a) Representation of the complete network diagram generated using the Biotapestry software (Longabaugh et al., 2005). (b) Schematic diagram describing the DBN which contains three tiers: I. TF …
Figure 4—figure supplement 1
Simulation of a single cell over time with different expression levels at the beginning.
The simulation rapidly stabilizes with characteristic TF expression levels irrespective of the starting conditions. (a) The expression levels of all 9 TFs are 0.2 at the start of the simulation. (b) …
Figure 5 with 2 supplements
The DBN recapitulates the consequences of TAL1 and LYL1 single and double perturbations as seen in vivo and in vitro.
Computational prediction of gene expression patterns for the nine TFs of interest after perturbation of TAL1 (a), LYL1 (b) or both (c). Deletion of TAL1 or LYL1 on their own has no major …
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Figure 5—source data 1
Raw and normalised data for the single cell gene expression experiments presented in this study.
1) TAL1 down-regulation (related to Figure 5 d), 2) PU.1 down-regulation (related to Figure 6 a), 3) GFI1B up-regulation (related to Figure 6b) and 4) AML-ETO9a perturbation (related to Figure 6 c)
- https://doi.org/10.7554/eLife.11469.047
Figure 5—figure supplement 1
Significance tests for the computational and experimental data after TF perturbations.
To determine statistical significance the Wilcoxon rank-sum test was used. Alterations to the expression profiles are indicated by the p-value; with statistically significance defined as follows: p <…
Figure 5—figure supplement 2
Histogram plots showing the gene expression distributions of all nine genes of the network for the perturbations presented in this study.
(a) LYL1 down-regulation; (b) TAL1/SCL down-regulation; (c) LYL1 and TAL1/SCL down-regulation; (d) PU.1 down-regulation; (e) GFI1B up-regulation; and (f) AML-ETO9a simulation.
Figure 6
The DBN captures the transcriptional consequences of network perturbations.
Left panel: Computational prediction of gene expression after perturbation of specific TFs. 1000 simulations were run for each perturbation to determine expression levels in a 'cell population' …
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Figure 6—source data 1
Summary of all computational simulations for perturbations of one or two TFs.
The results for a total of 162 simulations are shown. The data can be accessed using the embedded hyperlinks. The y-axes show the number of cells and the x-axes the relative expression level. Blue curves represent wild-type data and red curves represent perturbation data.
- https://doi.org/10.7554/eLife.11469.051
Author response image 1
