Histone deacetylases (HDACs) play important roles in cancer, and the FDA approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), is used for the treatment of advanced and refractory cutaneous T-cell lymphoma in patients with progressive, persistent, or recurrent disease. SAHA is part of more than 225 clinical trials. Inhibition of HDAC activity results in altered acetylation of core histones and other proteins that cause changes in gene expression, resulting in cell growth arrest and apoptosis. However, despite the massive investment the United States has made into the study of SAHA as a drug for human disease treatment, it is routinely stated that the mechanism of action of SAHA remains poorly understood. Recent findings from our lab suggest that SAHA and HDAC inhibitors actually have broader, noncatalytic effects on HDAC complexes. Here we will use innovative biochemical, quantitative proteomic, computational, and genomic technologies to further elucidate the mechanism of action of SAHA. In particular, we will analyze the SAHA induced disruption of a human HDAC protein interaction network and determine which fraction of SAHA induced gene expression changes can be accounted for by disrupting this network. In specific aim 1 we will complete our assembly of the protein interaction network and biochemically dissect the multiple complexes present in this network. Furthermore, we will develop novel computational approaches for protein interaction network assembly and determine the strength of interactions in this network. In specific aim two, we will finalize our analysis of the SAHA induced dynamics of the network and dissect the gene expression changes resulting from network disruption. In the third specific aim, we will carry out focused studies on a breast cancer cell line and compare these results to the standard cell line used in specific aims 1 and 2. By comparing and contrasting these results, we will determine the cell specific effects of SAHA on this human HDAC network. Upon completion of this application, we will have a detailed understanding in multiple cell types of the effects of SAHA on a histone protein interaction network and the resulting gene expression changes mediated by the disruption of this network. This information will yield novel insights into the mechanism of action of SAHA and facilitate future development of novel HDAC inhibitors.