UNIVERSITY OF MASSACHUSETTS
Identification of Transcription Factor Binding Sites in Human Promoters. Transcriptional regulation is a highly coordinated process in the human genome. A significant component of transcriptional regulation is the interaction between transcriptional factor proteins (TFs) and cis-regulatory DNA elements. Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-Seq) is a leading method for detecting binding sites of TFs across the human genome in living cells. The Encyclopedia of DNA Elements (ENCODE) public research consortium was established in September 2003 to identify all functional elements in the human genome. Projects currently underway within the ENCODE consortium are generating hundreds of ChIP-Seq data-sets for several human cell lines. Yet ChIP-Seq has a significant biological limitation: the functional consequences of the TF binding event must be determined by other means. A complementary experimental method utilizes high-throughput transient transfection promoter reporter assays in living cells. The advantage of this approach is that it directly measures the biological function of the promoter fragment. Measuring promoter activity using a transient reporter assay has the distinct advantage that the activity is localized precisely to the fragment cloned into the plasmid. Therefore, large scale reporter assays are ideal for validating the biological functions of promoters identified by ChIP-Seq. Reporter assays combined with site-directed mutagenesis have the additional advantage of pinpointing the functional TFBS at base pair resolution. We will conduct large-scale reporter assays to validate the biological function of promoters, with approximately half being detected by ChIP-Seq experiments and the remaining half being identified by the ENCODE Analysis Working Group (AWG). In this supplement, we specifically addresses the question of biological validation of regulatory regions identified in the ENCODE project.