File S1. ChIP/chip – detailed description
Two week old LEC1 over-expressing seedlings were treated with either DEX (or
ethanol as negative control) for 24h to promote the expression of LEC1-induced
genes. About 1g seedling leaf was vacuum infiltrated for 20min at 4°C in PBS (8mM
Na2HPO4, 2mM KH2PO4, 150mM NaCl, pH7.5) containing 1% v/v formaldehyde,
followed by a a 10min infiltration in 125mM glycine. The material was then washed
thoroughly in TBS (20mM Tris-HCl, 150 mM NaCl, pH7.5), transferred to 15mM TrisHCl, 2mM EDTA, 0.5mM spermin, 80mM KCl, 20mM NaCl, 15mM mercaptoethanol,
0.1% v/v Triton X100, pH7.5 (Dolezel et al. 1992), homogenized and passed through
a 50µm filter (Celltrics, Partec), and finally sedimented by centrifugation (20min at
3,000xg). The pellet was re-suspended in DOC (50mM Hepes, 140mM NaCl, 1mM
EDTA, 1% v/v TritonX-100, 0.1% sodium deoxycholate), 10% v/v glycerol, pH7.5,
incubated for 20min and centrifuged (12min at 12,800xg). The resulting pellet was
washed several times with the same buffer until the supernatant became colourless.
After a final wash in IP buffer (50mM Tris-HCl, 150mM NaCl, 1% v/v Triton X100,
pH7.9), the material was suspended in 800µl IP buffer and 400µl aliquots were
exposed to ultrasound to reduce the average fragment size of the chromatin to
500bp. To determine chromatin yield, 50µl aliquots were incubated overnight at 65°C
to disrupt DNA-protein cross-links, and the DNA was purified from the preparation
using a PCR purification kit (Qiagen, Hilden, Germany). The entire LEC1 sequence
(Arabidopsis thaliana1G21970) was cloned into the plasmid pET23a (Novagen) to
create a C-terminal fusion with a His-tag and a N-terminal T7 tag, and transferred into
E. coli BL21(DE3)Lys cells to obtain the heterologous expression of LEC1. The gene
product was purified by affinity chromatography on Ni-agarose (Qiagen), and used to
generate LEC1-specific polyclonal antibodies in rabbit. The rabbits were given three
injections of 800µg protein at 0, 28 and 38 days. Serum immunoglobulins were
concentrated by precipitation with 50% w/v ammonium sulphate. The precipitate was
dissolved in PBS and dialysed. The anti-LEC1 antibodies were purified by passing
through antigen-loaded CNBr-sepharose (Amersham). Protein concentrations were
determined by a Bradford assay. The anti-LEC1 antibody was tested using ELISA
and western blot analyses where it showed good specificity against recombinant
LEC1 protein in comparison to other recombinant proteins.
For each ChIP experiment, 10µg chromatin was combined with 2µg affinity-purified
anti-LEC1 antibody in 800µl IP buffer supplemented with DOC to a final
concentration of 0.1%. After a 2h incubation at 4°C, the antibody/chromatin complex
was captured by the addition of 50µl of protein A coated magnetic beads (Dynal) and
a further incubation of 2h. The beads were extracted from the supernatant with a
magnet, and washed for 5min at 4°C in a succession of 1ml volumes of: IP buffer,
then 50µM Tris-HCl, 0.5M NaCl, pH7.5, then 50µM Tris-HCl, 0.5M LiCl, 0.5% v/v NP
40, pH7.5, then 50µM Tris-HCl, 0.5M LiCl, 1% v/v NP 40, 1mM EDTA, 0.7% v/v
DOC, pH7.5, and finally 20mM TE (Tris-HCl, 1mM EDTA, pH7.5). The
antibody/chromatin complex was eluted from the beads by the addition of first 70µl,
and then a further 30µl 1% w/v SDS at 65°C. The combined eluate was mixed with
7µl 3M NaCl and incubated overnight at 65°C to abolish protein/DNA cross-linking.
The recovered chromatin fragments were purified using a PCR Purification kit
(Qiagen), and 1-2ng was treated with 5U Klenow polymerase (Fermentas, Vilnius,
Lithuania) and then ligated to 75pmol adaptor oligomers (oJW102 and oJW103, see
Odom et al. 2004) by treatment with 15U T4 ligase at 16°C for 12h. The ligation
reaction was terminated by heat inactivation (10min at 65°C), purified using a PCR
purification kit (Qiagen), and re-suspended in 45µl water. This preparation
represented the template for three 20µl PCR using 40 cycles of 95°C / 30s, 60°C /
60s and 72°C / 60s, in the presence of 10µM oJW102 sequence as primer, 200µM
dNTP, 2mM MgCl2 in a buffer of 10mM Tris-HCl, pH9.0, 50mM KCl, 0.1% v/v Triton
X100. The products of three replicate reactions were pooled and purified (Qiagen
PCR purification kit). Labelled probes were obtained by combining three parallel
random priming reactions produced with a Megaprime kit (GE Healthcare, Uppsala,
Sweden), 1ng amplified chromatin, and 50µCi [α-33P]dCTP per reaction.
For the large scale identification of precipitated and amplified chromatin fragments, a
macroarray was created based on 11,904 PCR products amplified from the upstream
regions of Arabidopsis thaliana genes (Benhamed et al. 2008). The amplicons were
purified on MinElute plates (Qiagen, Hilden, Germany) and re-suspended in 20µl TE.
Approximately 75% of the amplicons appeared as single fragments on agarose gels.
These were mixed with an equal volume of 1M NaOH, 5M NaCl and spotted in
duplicate onto a positively charged Biodyne B nylon membrane (Pall, Dreieich,
Germany) at 140 spots per cm2. The membranes were then washed in 0.5M NaOH,
1.5M NaCl for 5min, neutralized in 1.5M NaCl, 0.5M Tris-HCl, pH7.4 for 5min, crosslinked at 120mJ in a UV-Stratalinker 2400 (Stratagene, Cedar Creek, USA) and
baked for 30min at 80°C in a thermal oven. The arrays were pre-hybridized for 1h at
65°C in 0.5M sodium phosphate, pH7.2, 7% w/v SDS, 1% w/v BSA, 1mM EDTA,
10µg/ml sheared salmon sperm DNA, then labelled probe was added and the
hybridization reaction continued for 96h. Thereafter, the membranes were washed
twice in 0.2x SSC, 0.1% w/v SDS for 20min at 65°C and exposed to a Fuji image
plate. Hybridization signal was detected with a FLA 3000 phosphoimager (Fuji) at
50µm resolution and a 16 bit grey scale. Spot intensities were obtained by aligning
the pre-defined spotting pattern using ArrayVision software (GE Healthcare).
Four biological replicates were performed for LEC1_EtOH (control) and LEC1_DEX
(treatment) and all experiments were normalized by shifting the median of the log2
hybridization intensities to zero followed by a quantile normalization across the four
biological replicates (Bolstad et al. 2003). Double-spotting of intergenic regions on
the array enabled an internal control of normalized log2 hybridization intensities in
each replicate. Intergenic regions with a double-spot difference of less than one were
marked as reliable. For intergenic regions that were marked as reliable in three out of
four replicates the corresponding normalized log2 hybridization intensities of the
replicate that failed the double-spot test have been substituted by the mean log2
hybridization intensities of the three reliable experiments. Intergenic regions have
been excluded from all subsequent analyses if it they were considered as reliable in
less than three replicates. For each of the four replicates, the log-ratio of the mean
hybridization intensities in LEC1_DEX (treatment) to LEC1_ EtOH (control) has been
computed for each intergenic region to obtain a measure of IP-enrichment.
To identify putative intergenic target regions of LEC1, a Hidden Markov Model with
scaled transition matrices (SHMM) (Seifert et al. 2009) was applied to analyze the
log-ratios of the four replicates in the context of their chromosomal locations. Based
on that, intergenic regions of each replicate were ranked according to their probability
of representing a LEC1 target region. The 2,000 highest scoring intergenic regions of
each replicate were further analyzed to determine each intergenic region that was
contained in the scoring lists of at least three of four replicates. This resulted in 663
putative LEC1 intergenic target regions which were then subjected to further data
evaluation as described in Figure S2. Applying this threshold to a random selection of
2,000 promoter fragments per experiment produced about 34 hits.
After data normalization and statistical analysis, in total 663 promoter regions were
identified to be enriched in induced LEC1::GR seedlings after chromatinimmunoprecipitation with anti-LEC1 antibody. This number was corrected by taking
into account that several promoter amplicons, present on the SAP macroarray
represent divergent promoters depending on the direction of the adjacent genes (+/DNA strand). Theoretically, four genomic constellations of neighbouring genes are
conceivable
(head-to-head;
tail-to-tail;
head/tail-to-tail/head),
considering
the
promoter as head of the corresponding gene. Only three have to be taken into
account in ChIP data analysis. The intergenic region of genes in tail-to-tail
constellation does not represent a promoter region and is therefore not represented
on the SAP filter. The other three constellations were assumed to occur in equal
parts. The problem of divergent promoters had to be considered only for genes in
‘head-to-head’ constellation. In a head-to-head constellation ChIP does not
discriminate between a uni- or bidirectional promoter function of the intergenic region.
Additional experiments such as qRT-PCR are required to identify the true target
promoter. In total 360 LEC1::GR putative target genes have been found in head-tohead constellation with the adjacent genes. This fraction corresponds to 54,3% of all
LEC1::GR targets and exceeds the theoretically expected ratio of 33%. All putative
LEC1 target genes that occur in other genomic constellations than head-to-head
were considered as ‘candidate LEC1 targets’ (303 genes, Suppl. File 3).
For 65 head-to-head gene pairs both adjacent genes (130 of 663 genes) have been
identified as putative LEC1 target genes. For 4 gene pairs the expression of one of
both adjacent genes was found to be altered in response to DEX+ABA treatment in
the microarray experiment. These independently confirmed target genes were
considered as candidate targets. Another 230 putative LEC1 targets as identified by
ChIP/chip are located in head-to-head to its adjacent gene whereas this was not in
the list of candidates. These genes were classified according to the size of the
intergenic region. This size determines the overlap of spotted promoter fragments of
two adjacent genes. The bigger the intergenic region, the bigger are gene-specific
promoter regions near to TSS/ATG. Thus, for small intergenic regions, it is not clear
whether the LEC1 binding site is covered by both promoter fragments or if it is
located in gene specific parts of the spotted fragments. SAP promoter amplicons are
limited to a size of less than 2.5kB (Benhamed et al. 2008). Therefore a threshold
size of 3kB intergenic region was assumed to exclude an overlap in regions
containing putative LEC1 binding sites (500bp upstream of ATG). For 48 of these
gene pairs the intergenic region (from stop to ATG) was bigger than 3kb. The
corresponding 48 genes in the list of LEC1 target genes were considered as
candidate targets due to their large gene interspace. The remaining 216 head-tohead genes with an intergenic region of less than 3kb were supposed to share
important promoter regions with their neighbour gene. Although the promoter
fragments of 50 of these genes were supposed to show substantial overlap, the
adjacent gene was not found in the list of LEC1 targets. Among these 50 gene pairs,
for one gene pair one gene has been identified as putative LEC1 target by microarray
analysis revealing it as candidate target. Finally, for about 132 putative LEC1 targets
that are located in head-to-head in the genome the adjacent genes were not
represented on the SAP macroarray.
Benhamed, M., Martin-Magniette, M.L., Taconnat, L., Bitton, F., Servet, C., De
Clercq, R., De Meyer, B., Buysschaert, C., Rombauts, S., Villarroel, R.,
Aubourg, S., Beynon, J., Bhalerao, R.P., Coupland, G., Gruissem, W.,
Menke, F.L.H., Weisshaar, B., Renou, J.P., Zhou, D.X. and Hilson, P.
(2008) Genome-scale Arabidopsis promoter array identifies targets of the
histone acetyltransferase GCN5. Plant J., 56, 493-504.
Bolstad, B.M., Irizarry, R.A., Astrand, M. and Speed, T.P. (2003) A comparison of
normalization methods for high density oligonucleotide array data based on
variance and bias. Bioinformatics, 19, 185-193.
Dolezel, J., Cihalikova, J. and Lucretti, S. (1992) A HIGH-YIELD PROCEDURE
FOR ISOLATION OF METAPHASE CHROMOSOMES FROM ROOT-TIPS
OF VICIA-FABA L. Planta, 188, 93-98.
Seifert, M., Keilwagen, J., Strickert, M. and Grosse, I. (2009) Utilizing gene pair
orientations for HMM-based analysis of promoter array ChIP-chip data.
Bioinformatics, 25, 2118-2125.