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Our laboratory has been a leader in developing the technique of chromatin immunoprecipitation (ChIP) to study the function of mammalian transcription factors. Recently, we have extended our studies to allow a high-throughput, global analysis of transcription factor target genes by combining chromatin immunoprecipitation with genomic microarray hybridization (ChIP-chip assays). We began by developing the ChIP-CpG approach, using microarrays containing approximately 10,000 human or mouse CpG islands. More recently, the lab has worked in close collaboration with NimbleGen Systems, Inc. in Madison, WI, to analyze transcription factor binding sites and chromatin modifications on a genome-wide scale. Our current projects include the analysis of changes in chromatin structure that occur as normal cells transform to tumor cells and the identification of target genes of transcription factors such as OCT4 (a key regulator of stem cell self renewal), SUZ12 (a Polycomb Group protein involved in development and tumor progression), and the E2F/Rb family (key regulators of cell cycle progression). In addition, we are also developing analysis methods that enable the identification of consensus motifs and transcriptional regulatory modules from experimentally identified transcription factor binding sites.
genomics.ucdavis.edu/farnham
The Technique
A major focus of our research is the identification of binding sites for human transcription factors using ChIP-chip assays and genomic tiling arrays. Much of our research utilizes small tumor samples or primary cells that are difficult to grow in large quantities. When ChIP assays are performed using a small number of cells, it is necessary to amplify the samples to obtain enough DNA for hybridization to genomic arrays. We have found that the quality of the products is a major determinant of the quality of the ChIP-chip data. For many transcription factors, we were unable to obtain reproducible array data using the technique of ligation-mediated PCR (LM-PCR) to amplify the ChIP samples. This was due to unequal amplification of different regions in the experimental and control samples. However, we have found that the GenomePlex® whole genome amplification method provides excellent representation of the ChIP and input samples. The improvement in the quality of the products obtained using the GenomePlex protocol allows us to reproducibly identify binding sites that could not be distinguished from background noise using our previous protocols, enabling experimentation with factors and cell systems that are difficult to study.
Comparison of sample preparation methods for ChIP-chip assays, p. 577.
