Our lab is interested in the biochemical mechanisms by which the RNA polymerase II preinitiation complex (PIC) coordinates its functions with histone modification and remodeling enzymes necessary for transcription on chromatin. Work in previous funding cycles has established models for how the Mediator and TFIID co-activators coordinate with p300, Chd1, and SAGA, and how silencing proteins like HP1 and PRC1 influence this process on designer chromatin templates bearing specific histone modifications. Our most recent experiments have focused on how the H2AZ-H3.3 variant chromatin found at eukaryotic promoters regulates transcription. We have for the first time recreated transcriptional stimulation by acetylated variant chromatin in vitro, shown that it enhances binding of p400-Tip60 and Ino80, and will now pursue its mechanism. We employ the immobilized template assay, which allows us to capture PICs from HeLa and mouse embryonic stem cell extracts and analyze the functions of different PIC factors in vitro. The in vitro studies are accompanied by ChIP in a model cell-based system, and by analysis of genome wide data sets in the gene expression omnibus. In Aim 1, we will acquire knowledge of the role of enhanced binding of p400-Tip60 to variant chromatin on gene activation. In Aim 2, we will analyze binding and function of Ino80 on variant chromatin and the role Mediator plays in this process. Our emphasis is on how Ino80 responds to nucleosomal obstacles containing and lacking H2AZ and H3.3. Aim 3 examines the specific subunits dictating interaction of Mediator with TFIID, which form the core structure that controls binding of general factors, Pol II and the numerous chromatin factors necessary for initiation. Our results will provide a detailed mechanism of gene activation on chromatin using state of the art biochemical approaches.