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Blood, Vol. 114, Issue 2, 299-309, July 9, 2009
Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program
Blood Hoogenkamp et al.
114: 299
Supplemental materials for: Hoogenkamp et al
Files in this Data Supplement:
- Table S1. Primer sequences (5′–3′) (PDF, 81.3 KB)
- Figure S1 (JPG, 139 KB)
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(A) Example of a FACS profile of a day 3.75 differentiation culture. ES cells were differentiated into embryoid bodies and stained with a phycoerythrin (PE)-coupled antibody against Flk-1. Epiblast-like (−∕−) cells were sorted one day earlier. (B) Expression analysis of SCL, FLI-1, and C/EBPα mRNAs in the indicated cell types. For all other explanations, see legend of Fig. 1.
- Figure S2. Chromatin at Pu.1 and Csf1r cis-regulatory elements does not carry histone H3 K9 acetylation and histone H3K4 trimethylation in ES cells, mesodermal cells, and hemangioblasts (JPG, 163 KB)
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ChIP assays examining histone H3 K9 acetylation (A) and histone H3K4 trimethylation (B) at the indicated positions. 3T3 cells and PU.1 and CSF-1R expressing RAW264 macrophage cells were measured as negative and positive controls, respectively. (C) and (D): Comparison of H3K9 acetylation (C) and H3K4 trimethylation (D) at the indicated genes between Flk1+ Bry-GFP cells and unfractionated blast culture cells. Control and specific primer sequences are listed in Table S1 online. The results represent the mean values of two independent experiments analyzed in triplicate. Besides the amplicons shown we analyzed all genomic regions of Pu.1 and Csf1r that were either evolutionary conserved, marked by a DNaseI hypersensitive site in the appropriate cell types and/or have been shown to be of regulatory importance and did not find elevated levels of the indicated histone modifications (data not shown).
- Figure S3. Chromatin at Pu.1 and Csf1r cis-regulatory elements is not marked by trimethylation of H3 at lysine 27 (JPG, 74.1 KB)
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ChIP analysis using an anti-H3K27me3 antibody is shown for the indicated positions. Engrailed homologue 1 (En1), a known polycomb target gene, is used as a positive control. Control and specific primer sequences are listed in Table S1 online. The results represent the mean values of two independent experiments analyzed in duplicate.
- Figure S4. Csf1r and Pu.1 replicate early in ES cells and their derivatives (JPG, 173 KB)
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Replication timing analysis in sorted ES cell populations was carried out as described in Methods. α-Globin and X141 are used as controls for early and late replication respectively. Bars show locus abundance in each of 6 consecutive cell cycle fractions as determined by real-time quantitative PCR. The average and error of at least two experiments is shown.
- Figure S5. Chromatin priming in hemangioblasts does not involve extensive nucleosome remodelling at the Pu.1 promoter (JPG, 140 KB)
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Nuclei from the indicated cell types were digested with increasing amounts of MNase to yield predominantly mononucleosomal particles. DNA was purified and subjected to double-strand specific LMPCR. Samples that were digested to an equal extent as judged by agarose electrophoresis are marked by arrows.
- Figure S6 (JPG, 168 KB)
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(A) DNaseI accessibility assay analyzing the 3′ URE in the indicated cell types. For other explanations: see legend of Fig. 2. (B) Oct4 expression is downregulated during ES cell differentiation and DNaseI accessibility of Oct4 is reduced. DNaseI analysis was done using the same material as in Fig. 2. Signal intensities were measured across the displayed regions and were calculated relative to the rDNA control, with ES cells set as 1.
- Figure S7. PU.1, C/ebpα,and Csf1r expression and chromatin unfolding at Pu.1 cis-regulatory elements requires the transcription factor RUNX1 (JPG, 247 KB)
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(A and B) FACS-sorted hemangioblast cells from wt (Runx1+∕+), heterozygous Runx1+∕−, and knock-out Runx1−∕− ES cells were cultured for 4 days in blast culture medium (see Methods) and mRNA expression levels of Pu.1 (A), Csf1r (B), and C/ebpα (C) were determined during a 4 day time course of induction. (D) DNaseI in vivo footprinting assay examining the Pu.1 promoter and the URE in the indicated cell types (see legend of Fig. 2) including Flk1-positive hemangioblasts purified from wt (Runx1+∕+) and knock-out (Runx1−∕−) ES cells. (E) DNaseI accessibility at the Oct4 promoter in the hemangioblast population is not influenced by the presence or absence of RUNX1. Signal intensities were measured across the displayed regions and were calculated relative to the rDNA control, with ES cells or 3T3 cells set as 1 as indicated. The same material from the indicated cell types as used in (D) was amplified with primers specific for the Oct4 promoter. For other explanations: see legend of Fig. 2.
- Figure S8. Chromatin unfolding at Pu.1 cis-regulatory elements occurs in the absence of stable transcription factor binding (JPG, 175 KB)
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(A) DMS in vivo footprinting experiment analyzing the URE with the indicated cell types. Functional transcription factor binding sites are indicated on the right. Guanines displaying hyper-reactivity to DMS treatment in FACS purified CD41+ cells and macrophages are marked by black circles, protections from DMS-methylation are indicated by white circles. Only reproducible changes are marked. Remaining band intensity (at protected bands) or fold-enhancement (at hyper-reactive bands) is indicated next to binding sites (marked by arrows), with the G-reaction set as 1. No changes in DMS reactivity were seen in the other cell types examined or extended regions of both strands (data not shown).
- Figure S9. The Pu.1 promoter does not display stable DNA-protein interactions in hemangioblasts but the SCL promoter does (JPG, 194 KB)
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DMS in vivo footprinting experiment analyzing the Pu.1 promoter (A) and the Scl promoter (B). RAW: RAW264 cells, CD41+: CD41-positive cells sorted from blast cultures, MAC ES: Macrophages differentiated from ES cells, MAC: macrophages differentiated from mouse bone marrow.
- Figure S10. Pu.1 is a direct target of RUNX1 in blast cells (JPG, 112 KB)
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iRUNX1 cells were differentiated and induced with 0.1 µg/ml DOX as outlined and a ChIp assay was performed with day 4 blast cultures using antibodies against FLI-1, HA-tagged iRUNX1 and C/EBPβ. The values represent the mean values of at least two independent experiments.
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