A high-resolution interaction map of three transcriptional activation domains with a key coactivator from photo-cross-linking and multiplexed mass spectrometry.

Transcriptional activators play a central role in gene regulation by stimulating the assembly of the transcriptional machinery at a promoter in a signal-responsive manner. Because of the significant role that misregulated or malfunctioning transcriptional activators play in human disease, there has been a strong effort towards the identification of molecules that inhibit the ability of an activator to function through binding interactions with the transcriptional machinery. One difficulty associated with this strategy is that distinct transcriptional activators often share binding partners within the transcriptional machinery; thus, the design or discovery of molecules specific for a particular transcription factor is quite challenging. For example, the activators p53, c-Myc, and the viral oncoprotein E1A all interact with the coactivator and chromatin modifier TRRAP, yet it is not known if these activators utilize a shared binding surface for activation or if there are several distinct binding sites within the coactivator. Covalent cross-linking has been used successfully to identify coactivator binding partners of activators, but high-resolution analysis for binding-site identification has not been reported. A real challenge to highresolution analysis is the acid-rich composition of many eukaryotic activators, a characteristic that renders them difficult to analyze by standard cross-linking and mass spectrometric strategies. Herein we describe a multiplexed mass spectrometric strategy to produce a high-resolution map of the binding sites of transcriptional activators for the first time (Figure 1). We