Human Transcription factors (HTFs) and regulators that modify them are required to coordinate expression of the human genome/and their malfunction is implicated in causing cancers and other diseases in humans. The long-term goal of the proposed research here is to understand the molecular mechanisms governing the global transcription regulatory networks, as a prerequisite to developing therapeutic protocols to cure HTF- related cancers and diseases. We hypothesize that the many interactions between HTFs (monomers or dinners) and DNA motifs can be either convergent or divergent, and may be regulated by phosphorylation. As the first step towards this goal, the combined power of motif prediction algorithms and protein chip technologies will be applied to discover and validate novel motifs of HTFs. Regulation of the DNA-binding activity of HTFs will be examined by focusing on selected kinases in humans. Data will be integrated to initiate assembly of biochemical activity-based transcription circuitry. These goals will be achieved through the following four specific aims: (1) Fabricate HTF protein chips and determine the optimal assay conditions. 1003 purified HTFs and 96 heterodimers will be used to construct 400 HTF protein chips. (2) Profile motif- binding activities by using predicted motifs on the HTF protein chips. The association between a DNA motif and its binding factor will be determined on the HTF chips. (3) Determine the effects of MAPKs on motif- binding activity of HTFs. TFs that can be phosphorylated by MAPKs and related kinases will be identified in chip-based assays, and the effects of this modification on the DNA-binding activity of HTFs will be determined. (4) Validate the results from the above assays and build a map of transcription regulatory network. To validate the results, we plan to first apply a data integration step to generate robust hit lists for each type of high-throughput data set, followed by careful validation using conventional approaches (e.g., """"""""gel shift"""""""" and chromatin-IP assays for DNA motif validation). Using human cell lines, we will validate the kinase substrates in vivo: We expect that this project will fully establish a platform to inverstigate HTF activities and regulation, and to provide significant insight into the mechanisms of regulation of the human transcriptome and thus, help us to better understand the molecular mechanisms of related human diseases and cancer development and provide clues to improve public health.
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