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Supplementary materials and methods: Colony forming assay Colony forming assays were performed as described (Tagoh et al. 2002). CFU-mix medium, Methocult M3434 (Stem Cell Technologies), was used for myeloid colony assays. PreB cell mix, Methocult M3630 (Stem Cell Technologies) with additional cytokines (100 ng/ml SCF and 100 ng/ml VEGF), was used for lymphoid colony assay. Cells were seeded at the density of 1000 cells/ml and cultured in a moisture chamber under 5% CO2 at 37˚C for 8-10 days before scoring. Colonies grown under lymphoid conditions were picked for staining with CD19, NK1.1 and CD11b antibodies. Real Time-PCR mRNA expression analysis cDNA synthesis was carried out on DNaseI treated total RNA samples using oligo (dT) primers and M-MLV reverse transcriptase (Invitrogen). cDNA was subjected to Real Time quantitative PCR (ABI Prism 7700 sequence detection system, Perkin Elmer) with SYBR Green to measure the gene expression. Relative mRNA level was calculated using cDNA standard and the input was normalized against GAPDH expression. Chromatin immunoprecipitation assays Chromatin immunoprecipitation assays were performed essentially as previously published (Lefevre et al, 2003) with the following modifications. After 30min crosslinking with 1% formaldehyde at room temperature and cell lysis, pelleted nuclei were suspended into an equal volume of glycerol buffer (10mM HEPES, pH 7.4, 0.1mM EDTA, 25% glycerol, 2.5mM PMSF and protease cocktail inhibitor (Sigma, P-8340)). Nucleosomal material was generated by digesting with 500U/ml MNase (Roche) in 1xMNase buffer (25mM KCl, 4mM MgCl2, 1mM CaCl2 and 50mM Tris-HCl pH 7.4) for 10min at 37˚C. The reaction was stopped by adding EDTA(10mM). MNase-treated chromatin from 5x106 cells was used for each immunoprecipitation with acetyl H3K9 antibody (Upstate Biotechnologies), di-methyl H3K9 antibody (Abcam), tri-methyl H3K4 antibody (Abcam). Precipitated DNA was quantified using Real Time quantitative PCR with SYBR Green. Relative PCR signals for all primers were calculated as a signal ratio obtained with the specific antibody versus input. To correct for the efficiency of individual precipitation, signals were then normalized to those obtained with the GAPDH promoter (for acetyl K9 and tri-methyl K4) or ZFP37 promoter (for di-methyl K9). In vivo DMS footprinting In vivo DMS footprinting was performed exactly as previously described (Tagoh et al. 2002). Lesions in DMS-treated and piperidine-cleaved DNA were visualised by LM-PCR. In vivo DNaseI and MNase footprinting For in vivo DNaseI footprinting, the cell nuclei were prepared essentially described and treated with DNaseI as described in (Cockerill 2000) after crosslinking with 1% formaldehyde for 5 minutes. DNaseI accessibility was assayed by LM-PCR as described previously (Tagoh et al. 2002). For MNase footprinting DNA was extracted from the same chromatin prepared for ChIP assay after reversion of crosslinking by incubating at 65˚C for over night. DNA was first phosphorylated using T4 polynucleotide kinase (New England Biolabs) and then directly ligated to the linker, LP25-21 (Kontaraki et al., 2000), using T4 ligase (Promega). Ligated DNA was subjected to the primer extension with biotinylated primer for 12 cycles followed by LM-PCR amplification. DNA methylation assay Genomic DNA purified from individual cell types was digested with 10U of the methylation-sensitive restriction enzymes MaeII or HpaII, respectively. After inactivation of enzyme, digested DNA was precipitated and quantified by real-time quantitative PCR with SYBR green using primer sets spanning the enzyme recognition sites. Precise amounts of input DNA for each sample (in average 100-200 ng) were determined using a primer set, which did not 2 span an enzyme recognition site. The DNA amount was calculated relative to undigested standard DNA. Methylation in hematopoietic cells was calculated relative to the highly methylated DNA in fibroblasts. 3 4