BioProject

Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. More...

Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors. Overall design: Cells were transfected with siControl or siGREB1. The cells were treated with 10nM ICI 182780 for 24 hr to further deplete ER. 48 hr after transfection, the cells were treated with 100nM of Estrogen or vehicle for 6 hr and total RNA was collected from four biological replicates Less...