Description

This track displays genome wide localization of two transcription factors (USF1 and USF2) and acetylated histone H3 (H3ac) in a liver cell line (HepG2). ChIP was performed on three biological replicates and the samples were hybridized to Affymetrix arrays covering the human genome at an average resolution of 35 base pairs. This track shows average probe level intensities for each factor. The companion, Uppsala ChIP Sites track displays identified positive regions for each factor.

The raw data for this track is available at EBI ArrayExpress , as experiment E-TABM-314.

Methods

Chromatin immunoprecipitation was performed as previously described (Rada-Iglesias et al. 2005), but sonication conditions were optimized to obtain smaller fragments (approximately 300 bp), to further improve the resolution of our experiments. The antibodies against USF1 (H-86, sc-8983) and USF2 (C-20, sc-862) were from Santa Cruz biotechnology; anti-Histone H3 acetyl K9/14 (06-599) and normal rabbit IgG (12-370) were purchased from Upstate. Three completely independent biological replicates were performed for each antibody, obtaining the corresponding input as total genomic DNA reference. Hybridizations were performed using Affymetrix GeneChip Human Tiling 2.0R Array set (7 arrays set). Array data files for USF1, USF2, H3ac and IgG were normalized against corresponding Input arrays using Affymetrix Tiling Array Software (TAS) two-group normalization. An empirical Bayes algorithm was used to ensure that identified positive regions for USF1, USF2 and H3ac show low enrichment of IgG.

Verification

54 regions were selected to be analyzed by qPCR using new ChIP DNA obtained using USF1 (C20 and H86) and USF2 antibodies. Of those 54 regions, 6 were negative for both USF proteins and were used to set background/cut-off thresholds. For the remaining 48 regions all but one was positive both for USF1 and USF2 (with that unique negative region being different for USF1 and USF2). Furthermore, there was a high correlation (R=0,79-0,85) between the enrichments levels determined microarray hybridizations and qPCR.

Credits

These experiments were performed in the Claes Wadelius lab Dept. of Genetics and Pathology, Uppsala University, Sweden. Microarray hybridizations were performed at Affymetrix Inc., Santa Clara, USA. Data processing and statistical analysis was done at The Linnaeus Centre for Bioinformatics, Uppsala University, Sweden.

References

Rada-Iglesias A, Ameur A, Kapranov P, Enroth S, Komorowski J, Gingeras TR, Wadelius C. Whole-genome maps of USF1 and USF2 binding and histone H3 acetylation reveal new aspects of promoter structure and candidate genes for common human disorders. Genome Res. 2008 Mar;18(3):380-92.