Histone acetyltransferases (HATs) play a critical role in regulating gene expression of many cellular functions. Interestingly, the vision of histones being unique substrates for different HATs has recently changed, since HATs can also regulate essential functions such as protein turnover or cell cycle progression, via acetylation of non-histone proteins. HATs and their acetylation activity must have multiple physiological roles, which are still poorly understood. This is especially the case of GCN5 and PCAF, which are homologous HATs, but have different roles in vivo. Because a fine balance between acetylation and deacetylation regulates many cellular functions and the smallest change in this balance can lead to cancerogenesis, it is extremely important to understand the global acetylation function of HATs. In order to get more insights into functional role of HATs and acetylation in human cells in vivo, we propose to identify substrates of the human HATs, GCN5 and PCAF using systematic proteomic analysis. The targets of these HATs, either as recombinant enzymes or within their associated protein complexes, SAGA (Spt-Ada-Gcn5 acetylase) and ATAC (ADA Two-A containing Complexe), will be screened performing acetyltransferase assay on a human protein array containing more than 9000 proteins. Changes in acetylome will be also compared in between wild-type and cells lacking GCN5/PCAF using cutting-edge proteomic methodology such as Multidimensional Protein Identification Technology (MudPIT). Once the specific non-histone targets of these HAT complexes will be defined by the combination of these methods, we will carry out physiological studies to understand the in vivo regulatory role of these acetylation patterns determined by the SAGA and/or ATAC complexes. The success of this project will rely on the synergic interaction between the fellow’s unique expertise in proteomic studies and the knowledge of the host laboratory leader in the field of transcription regulation.