Download Supplementary materials and methods: Colony forming assay

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
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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.
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