developmental stage according to boyes et al. (2004): 1.02 plant treatment: EtOH antibody: anti-LEC1 antibody (non-commercial) antibody preparation: Antibodies specific for the transcription factor LEC1 were raised in rabbits. For antigen production, the whole gene fragment encoding LEC1 (AT1G21970) was cloned into the expression plasmid pET23a (Novagen) creating a C-terminal fusion with a His-tag and a N-terminal T7 tag. The LEC1 protein was produced in Escherichia coli BL21(DE3)Lys cells and purified via affinity chromatography on Ni-agarose (Qiagen). Rabbits were immunized three times (0d, 28d and 38d) with 800µg protein per injection. Immunoglobulins in the sera were enriched by precipitation with 50% saturated ammonium sulfate. The precipitate was dissolved in PBS, dialysed, and stored frozen at -20°C. The anti-LEC1 antibodies were purified via antigen-loaded CNBr-sepharose (Amersham). Protein concentrations were determined by Bradford assays. treatment time: 24h
Treatment protocol
Dexamethasone was added to a final concentration of 30µM and the isovolume of ethanol was added to control plants.
Growth protocol
Two weeks old 35S::LEC1::GR seedlings were treated with Dexamethasone or ethanol for 24 hours before cross-linking and chromatin isolation.
Extracted molecule
genomic DNA
Extraction protocol
About 1g seedling material was used for each chromatin preparation. For crosslinking plant material was vacuum infiltrated for 20min at 4°C in PBS (8mM Na2HPO4, 2mM KH2PO4, 150mM NaCl, pH7,5) plus 1% formaldehyde. The reaction was stopped by adding glycine (f.c. 125 mM) and continuing the infiltration for 10 min. All following steps were performed at 4°C and buffers were supplemented with a proteinase inhibitor cocktail (Roche). Seedlings were washed extensively with TBS and finally suspended in 15mM Tris-HCl, 2mM EDTA, 0.5mM spermin, 80mM KCl, 20mM NaCl, 15mM mercaptoethanol, 0.1% Triton X-100, pH7,5 (Dolezel et al., 1992). Seedlings were homogenized with a Potter homogenizer and passed through 50µm filters (Celltrics, Partec) and sedimented by centrifugation for 20min at 3000g. The pellet was resuspended in 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate (DOC), 10% glycerol, pH7,5, incubated for 20min and centrifuged for 12min at 12800g. The pellet was washed several times with the same buffer until the supernatant was colorless. After a final wash with IP buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, pH7.9) the chromatin was suspended in 800µl IP buffer. 400µl aliquots were treated with ultrasound to reduce the average fragment size of chromatin to 500bp. After centrifugation (12 min, 12000 g) the clear supernatant was stored at -80°C. To determine chromatin yield, 50µl aliquots were incubated overnight at 65°C for breaking DNA-protein cross-links, and purified using a PCR purification kit (Qiagen, Hilden, Germany). DNA concentration was determined by UV spectrometry using a NanoDrop 1000 (Peqlab,) photometer. For each immunoprecipitation 10µg chromatin were combined with 2µg of affinity purified anti-LEC1 antibody in 800µl of IP buffer supplemented with 0,1% DOC. After incubation for 2h at 4°C on a rotating wheel the antibody chromatin complex was captured on 50µl of protein A coated magnetic beads (Dynal) by continuing the incubation for 2h. The beads were separated magnetically from the supernatant and washed successively with 1 ml each of the following buffers: IP buffer; 50µM Tris-HCl, 0,5M NaCl, pH7,5; 50µM Tris-HCl, 0,5M LiCl, 0,5% NP 40, pH 7,5; 50µM Tris-HCl, 0,5M LiCl, 1% NP 40, 1mM EDTA, 0,7% DOC, pH 7,5, and TE buffer (20mM Tris-HCl, 1mM EDTA, pH 7,5). For each wash beads were incubated 5 min at 4°C on a rotating wheel and afterwards magnetically separated from the supernatant. The antibody-TF-DNA complex was eluted from the beads with 1% SDS at 65°C. Eluates from a first elution with 70 µl and a second elution with 30µl were combined, mixed with 7µl 3M NaCl, and incubated overnight at 65°C to reverse the cross-link between TF and DNA. Chromatin fragments were purified using the PCR Purification Kit (Qiagen) and their concentration determined using Picogreen (Invitrogen, Groningen, Holland) and a NanoDrop 3300 (Peqlab) fluorescence spectrophotometer. The control “input” DNA was treated identical. For amplification 1 – 2ng of chromatin fragments were blunted with 5U Klenow polymerase (Fermentas, Vilnius, Lithuania) and ligated with 75pmol adapters using 15U T4 ligase at 16°C for 12h. Adapters were obtained by mixing equal amounts of the oligonucleotides oJW102 (GCGGTGA¬CCCGGG¬AGATC¬TGA¬ATTC) and oJW103 (GAATTCAGATC) (Odom et al., 2004). Ligation reactions were terminated by heat inactivation (65°C, 10min), purified by reverse osmosis (PCR purification Kit, Qiagen, Hilden, Germany), and resuspended in 45µl water. Chromatin fragments were amplified for 40 cycles (95°C for 30s, 60°C for 60s, 72°C for 60s) using oJW102 as sole primer (10µM) in three separate reactions of 20µl (buffer: 10mM Tris-HCl, pH9,0, 50mM KCl, 0.1% (v/v) Triton X-100 containing 200µM dNTPs each and 2mM MgCl2). PCR products were pooled and purified (PCR purification Kit, Qiagen, Hilden, Germany).
Label
[α-33P]dCTP
Label protocol
Labelled probes were obtained by combining three parallel random priming reactions using the Megaprime Kit (GE Healthcare, Uppsala, Sweden), 1ng amplified chromatin, and 50µCi [α-33P]dCTP per reaction.
Hybridization protocol
Arrays were prehybridized for 1h at 65°C in Church buffer (0,5M sodium phosphate, pH7,2, 7% SDS, 1% BSA, 1mM EDTA, 10µg/ml sheared salmon sperm DNA) per cm2 of membrane in a rotating thermal oven. Labelled probes were added and hybridization continued for 4 days. Membranes were washed twice in 0,2x SSC, 0,1% SDS for 20 min at 65°C and then placed on a moist Whatman paper, wrapped in Saran wrap and exposed to a Fuji image plate for 10 days in a lead chamber.
Scan protocol
Signals were detected using a FLA 3000 phosphoimager at 50µm resolution and 16 bit grey scale (Fuji). Spot intensities were obtained by aligning the pre-defined spotting pattern using ArrayVision software (GE Healthcare).
Description
hybridization on membrane B
Data processing
Four biological replicates were performed and the group of LEC1_EtOH ChIP-chip experiments and the group of LEC1_DEX experiments were normalized independently. First, log-2-hybridization intensities were calculated from Dens - PSL / mm2 values (see raw data file) for each experiment. Then, the median of log-2-hybridization intensities of an experiment was centered to zero. Finally, quantile normalization was applied separately to both groups resulting in normalized log-2-hybridization intensities for each experiment (Bolstad et al., 2003). For quality control within an experiment each intergenic region was spotted twice on the macroarray resulting in two normalized log-2-hybridization intensities per intergenic region. A filter was applied to exclude each intergenic region from an experiment if the absolute difference in normalized log-2-hybridization intensities of both spots of this intergenic region was greater than one. Separately for both groups of experiments, missing normalized log-2-hybridization intensities of an excluded intergenic region were substituted by mean log-2-hybridization intensities of this region in other experiments if this region passed the filter in at least three other experiments. We used an inducible expression system and used DEX for induction and ethanol (as dissolvent for DEX) as a control treatment. The anti-LEC1 antibody was added to chromatin samples of both treatments and the results of the ethanol-treated sample as control values. [log2 ratio of normalized signal intensities of (EtOH/DEX)] data file is available on Series record.
Submission date
Jun 15, 2010
Last update date
Mar 31, 2012
Contact name
Astrid Junker
Organization name
Leibniz Institute for Plant Genetics and Crop Plant Research
