UNDERSTANDING HOW TWO RELATED HISTONE ACETYL TRANSFERASE CO-ACTIVATORS, SAGA AND ATAC, DIFFERENTIALLY REGULATE CHROMATIN DYNAMICS AND TRANSCRIPTION PROJECT SUMMARY Histone post translational modifications (PTMs) and the complexes that install them and read them, control many aspects of eukaryotic genome function including transcription, DNA repair and replication. As a result of histone PTM writer?s broad functions, they are essential in organismal development, aging and numerous diseases including cancer, heart disease, and even HIV integration. The majority of histone writers target the disordered N-terminal tail regions. Numerous histone writers have been identified and studied genetically, biochemically and structurally. Surprisingly, mechanistic studies are primarily limited to interactions between histone tail peptides and catalytic subunits, resulting in key gaps in understanding how histone readers/writers within their full native complexes interact and regulate chromatin structure, dynamics, accessibility and transcription. Recently, we developed the methods to purify biochemical quantities of endogenous human SAGA and ATAC complexes, which are related, but functionally distinct essential transcription co-activators. This allows us to quantitatively investigate how these two large multi-subunit complexes function relative to their common catalytic subunit, KAT2A, alone. Leveraging this, we recently found that (i) The endogenous SAGA and ATAC complexes acetylate histone octamers much more efficiently than the KAT2A acetyltransferase alone. (ii) The acetylation efficiency of endogenous SAGA and ATAC complexes are dramatically inhibited by unmodified nucleosomes relative to unmodified histone octamer. (iii) The SAGA and ATAC HAT modules acetylate histone H3K9 similarly relative to KAT2A alone, but the SAGA HAT module acetylates H3K9 in unmodified nucleosomes much more efficiently than the ATAC HAT complex or KAT2A alone. (iv) In mouse ES cells, deletion of the SAGA HAT module does not strongly affect global H3K9 acetylation, while deletion of the ATAC HAT module results in a significant reduction in overall H3K9 acetylation. These findings have led us to investigate the hypothesis that histone PTM readers/writers target chromatin properties through their accessory proteins to dynamically influence chromatin dynamics and accessibility. (1) Determine the chromatin properties that regulate reading and writing by the mammalian HAT complex SAGA. (2) Elucidate how the different subunits of the related mammalian HAT complexes, SAGA and ATAC, differentially control their targeting and chromatin modifying activity. (3) Determine the functional differences between SAGA and ATAC in mouse embryonic stem cells and during their differentiation. Together these aims provide a mechanistic and functional foundation for understanding of how two key transcriptional co-activators, SAGA and ATAC, differentially regulate chromatin dynamic and transcription.
Regulation of gene expression and genomic stability by the epigenetic factors, histone post translational modifications (PTMs) and the transcription co-activators multiprotein complexes, SAGA and ATAC, is a major focus in cancer epigenetics because of the reversible nature of epigenetic alterations. Recent advances in understanding epigenetic factors have led to the emergence of epigenetic therapy and the recent FDA approval of multiple epigenetic drugs. This proposal uses a combination in vitro biochemical, biophysical and single molecule methods, combined with in vivo genetic and cell biology approaches to reveal the molecular mechanisms behind the function of these chromatin epigenetic regulators. Our studies will provide molecular information for understanding drug resistance, tumorigenesis and will help designing new drugs and therapies. The proposed combination of different multidisciplinary and cutting-edge approaches, will ensure the success of the project. The project builds on the complementary expertise and knowledge already existing in the Poirier and Tora labs. In addition, our study on human SAGA and ATAC complexes will be directly applicable to a whole plethora of other large multiprotein transcription co-activator complexes. Thus, we anticipate that the results of our research will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription regulation.
