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Blood, Vol. 113, Issue 21, 5121-5124, May 21, 2009
The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters
Blood Bee et al.
113: 5121
Supplemental materials for: Bee et al
Files in this Data Supplement:
- Table S1. PCR and targeted mutagenesis primers (PDF, 41.8 KB)
- Table S2. Real-time PCR primers and probes (PDF, 44.1 KB)
- Figure S1. Comparison of Runx1 P2 core promoter sequences from zebrafish and mouse (JPG, 63.7 KB)
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(A) A 570bp genomic fragment spanning zebrafish core promoter sequences (UCSC danRer5). This fragment is located at the 3′ end of the 8.3 kb SacI-BamHI zebrafish P2 promoter fragment used in Lam et al.1 It contains 5 putative Gata, 7 Ets and 4 E-box motifs. This is reminiscent of the mouse Runx1 +23 enhancer, which was shown to be controlled by Gata, Ets and SCL (E-box) transcription factors in hematopoiesis.2,3 (B) Genomic sequence of the 205bp mouse P2 core promoter fragment used in our study (NCBIM36). This fragment contains 6 putative Ets motifs, but only one Gata motif and no E-box. Mouse and zebrafish P2 sequences do not align (negative data, not shown).
- Figure S2. Both the Runx1 P1 and P2 have active promoter marks in human primary CD133+ hematopoietic stem and progenitor cells (JPG, 70.2 KB)
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Genome-wide ChIP-Seq data from Cui et al.4 was mined for active promoter marks in the RUNX1 locus. High enrichment for both the P1 and P2 was found after ChIP for H3K4me3 modifications, indicative of active promoters. RUNX1 exons, the position of the promoters and the +23 enhancer are indicated.
- Figure S3. Negative control for Xgal staining (JPG, 143 KB)
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Sections through the E8 yolk sac (i), E10 vitelline artery (ii), E10 dorsal aorta (iii), and E12 fetal liver (iv) of a negative control embryos show the complete absence of background Xgal staining in our staining procedure. Photographs were taken using a Nikon Eclipse E600 microscope equipped with a 20x Nomarski objective and a Nikon DXM 1200c Digital Camera (Nikon, Tokyo, Japan) and processed using Adobe Photoshop (Adobe systems Europe, Uxbridge, United Kingdom). Scale bar = 100 µm.
REFERENCES
1. Lam EYN, Chau JYM, Kalev-Zylinska ML, et al. Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors. Blood. 2008;EPub ahead of print.
2. Nottingham WT, Jarratt A, Burgess M, et al. Runx1-mediated hematopoietic stem-cell emergence is controlled by a Gata/Ets/SCL-regulated enhancer. Blood. 2007;110:4188–4197.
3. Landry JR, Kinston S, Knezevic K, et al. Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver. Blood. 2008;111:3005–3014.
4. Cui K, Zang C, Roh TY, et al. Chromatin signatures in multipotent human hematopoietic stem cells indicate the fate of bivalent genes during differentiation. Cell Stem Cell. 2009;4:80–93.
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