Figure 1. 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 (Calero-Nieto et al., 2014) in HPC7 cells. Highlighted are all regions of the Erg gene locus that are acetylated at H3K27 and are bound by three or more TFs. Numbers indicate the distance (in kb) from the ATG start codon. (b) Summary of the identification of candidate cis-regulatory regions for all nine TFs and subsequent analysis in transgenic mouse assays. The inspection of the nine gene loci and the application of the selection criteria (≥3 TFs bound and H3K27ac) identified a total of 49 candidate cis-regulatory regions. The heatmap shows the binding pattern of the nine TFs to all candidate regulatory elements in HPC7 cells: green = bound, grey = unbound. Haematopoietic activity in E11.5 transgenic mice is indicated by the font color: black = active, red = not active. Grey indicates genomic repeat regions that were not tested in transgenic mice. Detailed experimental data corresponding to the summary heatmap can be found in Figure 1 and Figure 1—figure supplement 1–8. (c) Haematopoietic activity of the five candidate Erg cis-regulatory regions was determined in E11.5 transgenic mouse assays. Shown are X-Gal-stained whole-mount embryos and paraffin sections of the dorsal aorta (DA, ventral side on the left/top) and foetal liver (FL), sites of definitive haematopoiesis. Colour coding as in B.
DOI: http://dx.doi.org/10.7554/eLife.11469.003
Figure 1—source data 1. Number of PCR and LacZ positive transgenic embryos (E10.5–11.5) for each regulatory region.
elife-11469-fig1-data1.xlsx (12.2KB, xlsx)
DOI: 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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) Candidate regions were assayed for haematopoietic enhancer activity in mouse transient transgenic embryos. X-Gal stained whole-mount E11.5 embryos and paraffin sections of the dorsal aorta (DA; longitudinal section, ventral side on the left/top) and foetal liver (FL) are shown for the candidate cis-regulatory regions. Transgenic mouse data are not shown for previously published regions, but relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) Candidate regions were assayed for haematopoietic enhancer activity in mouse transient transgenic embryos. X-Gal stained whole-mount E11.5 embryos and paraffin sections of the dorsal aorta (DA; longitudinal section, ventral side on the left/top) and foetal liver (FL) are shown for the candidate cis-regulatory regions. Transgenic mouse data are not shown for previously published regions, but relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) All candidate regions were previously published. Relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) Candidate regions were assayed for haematopoietic enhancer activity in mouse transient transgenic embryos. X-Gal stained whole-mount E11.5 embryos and paraffin sections of the dorsal aorta (DA; longitudinal section, ventral side on the left/top) and foetal liver (FL) are shown for the candidate cis-regulatory regions. Transgenic mouse data are not shown for previously published regions, but relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) Candidate regions were assayed for haematopoietic enhancer activity in mouse transient transgenic embryos. X-Gal stained whole-mount E11.5 embryos and paraffin sections of the dorsal aorta (DA; longitudinal section, ventral side on the left/top) and foetal liver (FL) are shown for the candidate cis-regulatory regions.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) E10 embryos and cryosections of the DA (transverse; ventral down) and FL are shown. For the Runx1+204 region, a larger 12 kb fragment (chr16:92,620,915–92,631,936, mm9) was used for transient transgenesis, but similar results were obtained with the +204 fragment alone (data not shown). The +24 element was tested in conjunction with the +23 and did not change its tissue specificity (Bee et al., 2010). Preliminary data show that the +24 on its own does not mediate robust tissue specific expression of reporter genes. Transgenic mouse data are not shown for previously published regions, but relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering represents the direction and distance in kilobases from the start codon ATG (pro = promoter). (b) All candidate regions were previously published. Relevant publications are listed.
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 haematopoietic stem/progenitor cell line HPC7. Highlighted in pink are the candidate cis-regulatory regions which are bound by at least three of the nine TFs and showed H3K27 acetylation. The numbering is based on the distance (in kb) to promoter 1a. (b) All candidate regions were previously published. Relevant publications are listed.