Elucidating Protein: DNA Complex by Oligonucleotide DNA Affinity Purification
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.
Key wordsTranscription activation complex Oligonucleotide DNA affinity purification Transcription factor NFAT Transcription co-factors Protein kinases
The authors would like to acknowledge the past and current members of the Chow laboratory for their input, discussion, and trouble shooting of the protocol.