The posttranslational acetylation of histone proteins is a crucial regulator of chromatin structure and function. Furthermore, global abnormalities in histone acetylation patterns occur early during the course of tumorigenesis, suggesting they may be relevant steps in the transformation process. However, the epigenetic role of particular histone acetylation events, and the mechanisms leading to their dysregulation in diseases, is poorly understood. Further elucidation of the precise role of histone acetylation will require new, orthogonal tools that allow researchers to study single acetylation events in vivo. The proposed research will employ in vitro evolution methods to develop ribozymes that site-specifically acetylate histone proteins in cells. Histone H4 lysine 16 (H4K16) will be the initial target due to the potent effect of this residue on the structure and function of chromatin. Peptides derived from the N-terminal tail of histone H4 will serve as in vitro substrates during evolution, and ribozymes will be selected based on their ability to acetylate these peptides at lysine 16. Individual ribozyme clones from the final round of selection will be assayed for acetyltransferase activity in the context of free histone proteins, nucleosome core particles, and nucleosomal arrays. Competent ribozymes will be engineered for increased stability against cellular degradation and expressed in cells using a cassette base on the high copy number U6 snRNA promoter. In vivo acetylation of H4K16 will be verified by Western blot and mass spectrometry analysis of endogenous histone proteins isolated from cells expressing histone acetyltransferase (HAT) ribozymes. In addition, a comprehensive transcriptome analysis will be conducted on cells expressing HAT ribozymes. Ribozymes developed using these methods will allow researchers to study H4K16 acetylation, as well as the enzymes associated with this modification, with greater analytical precision compared to approaches that are currently achievable. Such studies may lead to new diagnostics and therapies for cancer. In addition, the proposed selection strategy may eventually be used to evolve HAT ribozymes that target other histone residues or even non- histone proteins.
Global abnormalities in histone acetylation patterns appear early and accumulate during the course of tumorigenesis and are a common hallmark of cancer cells (1). A detailed explanation for these phenomena (and insight into possible treatment options) is impeded by the limited availability of tools for studying histone acetylation (2,3). Th proposed research aims to develop a novel tool that will allow researchers to study histone acetylation with greater analytical precision compared to approaches that are currently achievable. !
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