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Blood, Vol. 112, Issue 5, 1673-1682, September 1, 2008
Stem cell–specific epigenetic priming and B cell–specific transcriptional activation at the mouse Cd19 locus
Blood Walter et al.
112: 1673
Supplemental materials for: Walter et al
Files in this Data Supplement:
- Table S1. Sequence of oligonucleotides used in the experiments (PDF, 974 KB)
- Figure S1. DNaseI hypersensitive sites mapping at the mouse Cd19 locus (JPG, 28.8 KB)
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(A) A map indicating exons, introns, transcription start sites, and conservation (http://genome.ucsc.edu/). The positions of restriction enzyme sites (EcoRI, BlpI, and ScaI) and probes used in (B) are indicated as vertical lines and boxes with arrows above the map. (B) Summary of DHSs in this region. Grey bars represent the position of DHSs which are either weak or not lymphoid specific. Block arrows represent the position of strong hypersensitive sites at the promoter and −2 kb. The intensity is indicated by the arrow’s width. Dashed arrows at the bottom indicate the position of the promoter and enhancer.
- Figure S2. Induction of active form PAX5 by OHT (JPG, 77.7 KB)
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(A) Cd19 expression was after OHT induction of PAX5 in Pax5-ER cells. Pax5-ER cells were stimulated with 1 µM OHT for various period of time. Gene expression was measured and the relative expression was determined as described in Materials and Methods. The data represent the mean of two independent experiments. (B) PAX5 recruitment was analyzed by ChIP assays in Pax5-ER cells induced with OHT. The enrichment was measured at the promoter and a downstream region at +10 kb. The value for RNAP II was normalized against the enrichment at rRNA promoter. Data are representative of two independent experiments performed in triplicate. (C) DMS in vivo footprinting assays were performed at the Cd19 promoter in pro-B cell lines. WT: wild type pro-B cells, −/−: Pax5−/− pro-B cells, Pax5-ER + OHT: Pax5-ER cells induced with OHT. See Figure 2 legend for description of the symbols.
- Figure S3. Primary cell purification from mouse bone marrow (JPG, 91.1 KB)
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(A) Surface marker profile of cell purification from mouse bone marrow cells.
Multicolour staining was performed on the bone marrow cells following the removal of erythroid, myeloid and T-cell lineages. mRNA expression of Cd19 (B) and Pax5 (C) was measured by using quantitative real time PCR. Relative expression was determined as described in Materials and Methods.
- Figure S4. Determination of the level of CpG methylation (JPG, 157 KB)
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(A) The position of CpGs (boxed), putative transcription factor binding sites (horizontal lines) and major transcription start sites (hooked arrows) are indicated at the DNA sequence of promoter (left panel) and enhancer (right panel) regions. (B) Examples of gel images of several sequencing reactions. PCR products amplified on sodium bisulfite-modified DNA were subjected to the dideoxy sequencing reaction. The position of CpGs (CpG 3 − 6 at the promoter and CpG 1 − 5 at the enhancer) are indicated on the right hand side of the image. Cells analysed are listed below the images. Band intensities of T and C corresponding to certain CpGs were measured by using Quantity One® software and plotted in Figure 5.
- Figure S5. Quantification of the level of DMS modification at −2036 in the enhancer (JPG, 29.4 KB)
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The band intensity of in vivo DMS footprinting (Figure 6A) was measured using Quantity One® software. The value was normalised against the total lane intensity and values for G reaction were set as one.
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