Description

This track shows the map of -log10(P-value) for ChIP-chip using DNA from immunoprecipitated chromatin from either human HelaS3 (cervix epithelial adenocarcinoma), GM06990 (lymphoblastoid) or K562 (myeloid leukemia-derived) cells hybridized to maskless photolithographic arrays. The arrays consist of 50-mer oligonucleotides tiled with 12-nt overlaps covering most of the non-repetitive DNA sequence of the ENCODE regions. Chromatin immunoprecipitation was carried out for each experiment using antibodies against the following targets: BAF155, BAF170, INI1/BAF47, c-Fos, c-Jun, TAF1/TAFII250, RNA polymerase II, histone H4 tetra-acetylated lysine (H4Kac4), histone H3 tri-methylated lysine (H3K27me3), STAT1, nuclear factor kappa B (NFKB) p65, SMARCA4/BRG1, SMARCA6 and NRSF. Additionally, HeLa S3 cells immunoprecipitated with STAT1 were pre-treated with interferon-alpha and HeLa S3 cells immunoprecipitated with NFKB antibody were pre-treated with tumor necrosis factor-alpha (TNF-alpha) (see table below).

This track shows the combined results of three or four multiple biological replicates. For all arrays, the ChIP DNA was labeled with Cy5 and the control DNA was labeled with Cy3. These data are available at NCBI GEO (see table below for links), which also provides additional information about the experimental protocols.

Display Conventions and Configuration

The subtracks within this annotation may be configured in a variety of ways to highlight different aspects of the displayed data. The graphical configuration options are shown at the top of the track description page, followed by a list of subtracks. To display only selected subtracks, uncheck the boxes next to the tracks you wish to hide. For more information about the graphical configuration options, click the Graph configuration help link.

Methods

The data from replicates were quantile-normalized and median-scaled to each other (both Cy3 and Cy5 channels). Using a 1000 bp sliding window centered on each oligonucleotide probe, a signal map (estimating the fold enrichment [log2 scale] of ChIP DNA) was generated by computing the pseudomedian signal of all log2(Cy5/Cy3) ratios (median of pairwise averages) within the window, including replicates. Using the same procedure, a -log10(P-value) map (measuring significance of enrichment of oligonucleotide probes in the window) for all sliding windows was made by computing P-values using the Wilcoxon paired signed rank test comparing fluorescent intensity between Cy5 and Cy3 for each oligonucleotide probe (Cy5 and Cy3 signals from the same array). A binding site was determined by thresholding oligonucleotide positions with -log10(P-value) (>= 4), extending qualified positions upstream and downstream 250 bp, and requiring 1000 bp space between two sites. Top 400 sites are retained.

Verification

ChIP-chip binding sites were verified by comparing "hit lists" generated from combinations of different biological replicates. Only experiments that yielded a significant overlap (greater than 50 percent) were accepted. As an independent check (for maskless arrays), data on the microarray were randomized with respect to position and re-scored; significantly fewer hits (consistent with random noise) were generated this way.

Credits

These data were generated and analyzed by the labs of Michael Snyder, Mark Gerstein and Sherman Weissman at Yale University.

References

Cawley S, Bekiranov S, Ng HH, Kapranov P, Sekinger EA, Kampa D, Piccolboni A, Sementchenko V, Cheng J, Williams AJ et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 2004 Feb 20;116(4):499-509.

Euskirchen G, Royce TE, Bertone P, Martone R, Rinn JL, Nelson FK, Sayward F, Luscombe NM, Miller P, Gerstein M et al. CREB binds to multiple loci on human chromosome 22. Mol Cell Biol. 2004 May;24(9):3804-14.

Martone R, Euskirchen G, Bertone P, Hartman S, Royce TE, Luscombe NM, Rinn JL, Nelson FK, Miller P, Gerstein M et al. Distribution of NF-kappaB-binding sites across human chromosome 22. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12247-52.

Quackenbush J. Microarray data normalization and transformation Nat Genet. 2002 Dec;32(Suppl):496-501.