Identification of Small Binding Motifs to the Transcription Factor CP2 using the High-throughput Phage Display

초록

Although CP2 is a ubiquitous transcription factor expressed in most of tissue, it also involves in the tissue-specific or stage-specific transcriptional activation of target genes, such as globin in red blood cells, cytokine IL-4 in helper T cells, cytochrome P450scc in placenta, and SAA3 (serum amyloid A3) in liver. It suggests that this specificity is regulated by interactions with other cellular partner proteins or by specific modifications of the CP2 protein. To identify the factors that modulate CP2 activity or directly participate in CP2-mediated transcriptional activation, we screened a phage display library and isolated a bunch of CP2 binding peptides. Through searching protein databases, we identified several putative CP2-binding proteins, including YY1 that was already known to interact with CP2, a protein inhibitor of activated STAT1 (PIAS1), and members of serine/threonine kinase family. We confirmed the binding of candidate proteins to CP2 in vivo and in vitro, and also determined their CP2 binding affinity as well as the binding domains in the CP2 protein. Our results suggests that many cellular proteins dynamically interact with CP2 and these specific protein-protein interactions modulate the transcriptional activity of CP2 by affecting assembly of several multifunctional protein-protein interactions and/or specific modification of the CP2 protein. We are currently testing functional significance of these CP2 interacting proteins on the sequence-specific DNA binding and transcriptional activation of CP2 by employing constructs containing CP2 binding sites in the mouse -globin gene promoter as a model.