## Description

This track displays the results of ENCODE region-wide localization for three transcription factors (HNF-3b, HNF-4a and USF-1) and acetylated histone H3 (H3ac). The heights of the peaks in the graphical display indicate the ratio of enriched non-amplified DNA to input DNA.

The data for each of the transcription factors and H3ac are displayed in individual subtracks. The analysis cut-off threshold is indicated in each subtrack by a horizontal line. Tentative binding sites (TBSs) in spots passing the cut-off are displayed in a separate subtrack, ChIP-chip (HepG2) Sites. These sites are numbered corresponding to the ranking of spots based on enrichment ratios. Each TBS is assigned a value indicating how often it was found in separate BioProspector software runs for the prediction of TBSs (e.g. 1000 indicates that a TBS was found in ten out of ten runs).

The raw data for this track is available at EBI ArrayExpress, as experiment E-MEXP-452.

## Methods

Chromatin from HepG2 cells was cross-linked with formaldehyde and sonicated to produce DNA fragments of size 0.5-2 kb. Chromatin was precipitated using antibodies against HNF-4a, HNF-3b, USF-1 or H3ac. DNA from a single ChIP reaction was labeled with Cy5, and a fraction of the total input was labeled with Cy3. There was no amplification of the ChIP DNA or the input DNA prior to this step to avoid introducing bias.

This DNA was combined and hybridized to PCR-based tiling path ENCODE arrays. Most array elements were printed only once on the slide, but X-chromosomal regions (ENm006 and ENr324) were printed in duplicate. There were approximately 19,000 spots/slide. The array provided about 75% coverage of the ENCODE regions.

Spots flagged as bad by the image processing step were removed; those that remained were normalized. The average log2 ratio was calculated for spots that were replicated on the array. A log odds score for differential enrichment with the negative control was calculated using an empirical Bayes method. There were four log odds scores for each spot, one for each antibody. If this score was greater than 0 and the log2 ratio was greater than 1.25 (indicative of a strong positive signal), based on at least 2 replicates, the spots were considered to be enriched.

Binding sites were identified using the BioProspector software. Because the software is non-deterministic, different runs may produce different results for the same data. Predictions consistent across many runs are more likely to be correct; therefore, the analysis was repeated, keeping all binding sites occurring in each top-scoring motif to generate a set of candidates. TBSs present in at least five out of ten runs were selected. Further method details are described in Rada-Iglesias et al. (2005).

In the graphical display, overlapping sequences were removed by changing the start position of downstream spots to generate a continuous track. To give each track a comparable scale, the values for the most enriched spots were lowered to 15. Spots deemed as false positives, when compared to a no antibody ChIP-chip experiment, were assigned a value of 0.

## Verification

A negative control was done using no antibody for the ChIP-chip to reduce the number of false positives. Three independent biological replicates were performed for each antibody; three negative control ChIPs were also analyzed. Semi-quantitative PCR was used to verify enrichment in at least ten positive spots for each antibody.

## Credits

These experiments were performed in the Claes Wadelius lab. The statistical analysis was done at the Linnaeus Centre for Bioinformatics at Uppsala University. Microarrays were produced at the Sanger Institute.

## References

Rada-Iglesias, A. et al. Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays. Hum Mol Genet. 14, 3435-3447 (2005).