Description
This track shows regions that co-precipitate with antibodies against
each of 4 factors in all ENCODE regions, in retinoic-acid stimulated
HL-60
(leukemia) cells harvested after 0, 2, 8, and 32 hours, and in a fifth
factor tested in
ME-180 cervical carcinoma cells.
Clustered sites are shown in separate subtracks for each antibody:
- H4Kac4 (HisH4) - Histone H4 tetra-acetylated lysine
- H3K9K14ac2 (H3K9K14D) - Histone H3 K9 K14 Di-Acetylated
- Pol2 - RNA Polymerase II (8WG16 ab against pre-initiation complex form)
- p63_ActD - p63, in actinomycin-D treated ME-180 cells
- p63_mActD - p63 in untreated ME-180 cells
Retinoic acid-stimulated HL-60 cells and ME-180 cells (actinomycin-D treated
or untreated) were harvested and
whole cell extracts (control) were made. An antibody was used to
immunoprecipitate bound chromatin fragments (treatment). DNA was
purified from these samples and hybridized to Affymetrix ENCODE
oligonucleotide tiling arrays, which have 25-mer probes tiled
every 22 bp on average in the non-repetitive ENCODE regions.
Data for all biological replicates
can be downloaded from Affymetrix in
wiggle,
cel, and
soft formats.
Display Conventions and Configuration
The subtracks within this composite annotation track
may be configured in a variety of ways to highlight different aspects of the
displayed data. The graphical configuration options for the subtracks
are shown at the top of the track description page, followed by a list of
subtracks.
For more information about the graphical configuration options, click the
Graph
configuration help link.
Color differences among the subtracks are arbitrary. They provide a
visual cue for finding the same antibody in different timepoint tracks.
Methods
Three independent biological replicates were generated and hybridized
to duplicate arrays (two technical replicates). Reproducible enriched regions
were generated from the signal, by first applying a cutoff
of 0.693(ln(2)=0.693) to the signal estimate, a maxgap and minrun of 500
and 0 basepairs respectively, to each biological replicate.
Since each region or site can comprise of more than a single probe,
a median based on the distribution of log transformed P-values
was computed per site for each of the respective replicates.
These seed sites were then ranked individually within each of the replicates.
If a site was absent in a replicate the maximum or worst rank of the
distribution was assigned to it.
The following three values were computed for each
site by combining data from all biological replicates:
- average of all ranks computed among biological replicates
- sum of all pairwise differences in these ranks computed among biological
replicates
- a combined P-value, using a chi square distribution,
across all replicates
A final signal estimate based filter was applied, where sites with median
signal estimate of at least 0.693/(total number of individual replcates)
were considered. This was to ensure that if a site was not
detected consistently in all replicates but was detected at a significant
signal level in a subset of the replicates its detection level would
be weighted accordingly in the final selection of sites.
The final sites were selected when all of the above three metrics were
relatively low, where "low" corresponds to the top 25
percentile of the distribution.
Verification
Using the P-values from the biological replicates, all pairwise
rank correlation coefficients were computed among biological
replicates. Data sets showing both consistent pairwise correlation
coefficients and at least weak positive correlation across all pairs
were considered reproducible.
Credits
These data were generated and analyzed by the Gingeras/Struhl
collaboration with the Tom Gingeras group at
Affymetrix and
Kevin Struhl's group at Harvard Medical School.
References
Please see the
Affymetrix Transcriptome site for a project overview and
additional references to Affymetrix tiling array publications.
Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P.
A comparison
of normalization methods for high density oligonucleotide array data based
on variance and bias.
Bioinformatics 19(2), 185-193 (2003).
Cawley, S., Bekiranov, S., Ng, H. H., Kapranov, P., Sekinger,
E. A., Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J.,
Williams, A. J., et al.
Unbiased mapping of
transcription factor binding sites along human chromosomes 21 and 22 points
to widespread regulation of noncoding RNAs.
Cell 116(4), 499-509 (2004).
Yang A, Zhu Z, Kapranov P, McKeon F, Church GM, Gingeras TR, Struhl K.
Relationships between p63 binding, DNA sequence, transcription
activity, and biological function in human cells.
Mol. Cell. 24(4), 593-602 (2006).
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