Polynucleotide High Performance Affinity Chromatography

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DESCRIPTION (provided by applicant): The goal is to improve the performance of the DNA-affinity chromatography of transcription factors and to increase understanding of genetic regulation. Many of the transcription factors (TFs; e.g., c- jun, c-myc) were originally discovered as oncogenes and genetic regulation is important to the understanding of genetic disease. The relevance to human health and well-being is clear. We will: 1. Develop a novel and innovative method for characterizing promoters. The way promoter structure is now studied is to identify each response element contained within its sequence, use the electrophoretic mobility shift assay to detect whether the isolated element is in fact bound by a nuclear protein, and then use DNA-affinity chromatography and other methods to purify the proteins, one-at-a-time. For complex promoters, this linear approach is tedious and prolonged. Promoter Trapping was developed in the last funding period and has the potential to provide a completely novel way to characterize a promoter; mapping and identification of all transcription factors that bind in a few steps using gel blots. We have recently combined 2DGE with southwestern blotting and on- blot digestion to allow the sensitive detection of TFS and allow mass spectrometry characterization directly from the blot. Combined with promoter trapping, these promoter complexes can likely be analyzed from a few gel blots. We will determine if this new approach can speed research progress. 2. Novel 3-dimensional gel electrophoresis (3DGE) methods will be developed to solve intractable TFs. Three new 3DGE methods will be developed to further separate the proteins obtained from systematic oligonucleotide trapping (SOT) and promoter trapping (PT). The first dimension in each case relies on specific DNA binding electrophoresis, then a second and third dimension of pI and SDS-PAGE. These new techniques will resolve any intractable transcription factor identifications. 3. Alternative trapping methods will solve problems with current trapping technology. SOT has been proven to be the most powerful techniques available for purifying transcription factors but it is not perfect. The ends of the oligonuleotide used for trapping are binding DNA repair proteins as contaminants which can obscure identification. We will develop alternatives which use either thiol-disulfide chemistry or alternatively, immobilized metal affinity chromatography, to solve this problem. The result of the proposed studies will be truly innovative and transformative ways to study genetic regulation. These experiments will require four years. PUBLIC HEALTH RELEVANCE: Many oncogenes are transcription factors, showing the relevance of transcription factors to cancer and also the importance of transcription factors to cell division and other biological processes. To understand genetic disease and genetic regulation, the function of transcription factors must also be understood. Here, we improve the purification and characterization of these important proteins.