Description

ChIP analysis was performed using antibodies to E2F1, c-Myc, TAFI and PolII in HeLa, GM06990 and/or HelaS3 cells. E2F1 and c-Myc protein are transcription factors related to growth. E2F1 is important in controlling cell division, and c-Myc is associated with cell proliferation and neoplastic disease. TAFI is a general transcription factor that is a key part of the pre-initiation complex found on the promoter. PolII is RNA polymerase II.

For E2F1 and c-Myc, three independently crosslinked preparations of HeLa cells were used to provide three independent biological replicates. ChIP assays were performed (with minor modifications which can be provided upon request) using the protocol found at The Farnham Laboratory. Array hybridizations were performed using standard NimbleGen Systems conditions.

For TAFI and PolII, crosslinked cells were officially supplied by the ENCODE Consortium (for reference, see The Human Genetic Cell Repository). Hence, this data may be compared to other tracks using this exact source of cells. (Note that this is different from the E2F1 and c-myc subtracks — those Hela cells were grown in the Farnham lab.) ChIP-chip and amplification procedures are according to standard protocols available in detail from the Farnham Lab website. Whole Genome Amplification (WGA) was used for these samples. Array processing was performed by NimbleGen, Inc. The supplied array data is the result of three biological replicates in each case.

Display Conventions and Configuration

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Methods

Ratio intensity values (antibody vs. total) for each of three biological replicates were calculated and converted to log₂. Each set of ratio values was then independently scaled by its Tukey biweight mean. The three replicates were then combined by taking the median scaled log₂ ratio for each oligo.

Verification

For E2F1, primers were chosen to correspond to 13 individual peaks. PCR reactions were performed for each of the 13 primer sets using amplicons derived from each of three biological samples (39 reactions). The PCR reactions confirmed that all of the 13 chosen peaks were bound by E2F1 in all three biological samples.

For PolII, simple verification of the ChIP sample was performed at a known positive target (the promoter for POLII) and known negative target (the DHFR 3' UTR region). Quantitative PCR verifications of sites are in progress.

Credits

These data were contributed by Mike Singer, Kyle Munn, Nan Jiang, Xinmin Zhang, Todd Richmond and Roland Green of NimbleGen Systems, Inc., and Matt Oberley, David Inman, Mark Bieda, Shally Xu and Peggy Farnham of Farnham Lab.

Reference

Bieda M, Xu X, Singer MA, Green R, Farnham PJ. Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome. Genome Res. 2006 May;16(5):595-605.