CTIP-Mediated Transcriptional Repression
Speaker: Cortny Huddleston
COUP-TF interacting protein (CTIP) interacts with a complex of proteins that repress transcription. The mechanism underlying CTIP-mediated transcriptional repression is unknown, however CTIP does not interact with proteins responsible for classical pathways of repression. The lack of interaction with key proteins of this classical pathway does not exclude the possibility that CTIP repression is mediated by this pathway, but it does call for an investigation into other, perhaps novel, means of repression. In vitro protein interaction studies between CTIP and TATA binding protein (TBP), a key DNA binding protein of the transcriptional machinery, show that CTIP and TBP interact in vitro with high affinity. If these proteins interact in cells, it may suggest that transcriptional repression mediated by CTIP is directed at the transcriptional machinery.
A single assay will not determine solid evidence for interaction, so several techniques must be used. First, living cells must successfully accept a transfection, a process in which the cell accepts and incorporates DNA encoding CTIP or TBP or both into the cell. The proteins are then extracted from the cells and the interaction can be detected by coimmunoprecipitation, a process that detects one or more single proteins among a complex of proteins. If CTIP is immunoprecipitated out of a complex and TBP interacts with it, TBP can then be detected by a second antibody, specific to TBP. The localization of CTIP and TBP can also be detected in transfected cells by detecting fluorescent antibodies bound to CTIP and TBP in whole cells. Once the interaction between CTIP and TBP is found, the precise area of the protein interaction can be detected using cloned fragments of one of the proteins. If a mutant of TBP interacts with the whole CTIP protein, determined by the same assays mentioned above, the TBP fragment would be refined until the exact amino acid sequence of interaction is known.