title: Elucidating protein: DNA complex by oligonucleotide DNA affinity purification.
abstract: Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.
authors: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA.
authors: Teddy T C
authors: TT
authors: Yang
authors: Chi-Wing
authors: CW
authors: Chow
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism
chemical_list: D050778:NFATC Transcription Factors; D011506:Proteins; D004247:DNA; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038744:Ribosomal Protein S6 Kinases, 90-kDa