The proposed research in this training plan will focus on studying the effects of cis-trans proline isomerization on chromatin structure and function. The physiological state of DNA in eukaryotic cells occurs in the form of chromatin. As a result, access to the genome for fundamental cellular processes such as cell division, differentiation, and homeostasis must occur at the chromatin level. This has led to a widespread investigation of chromatin regulatory mechanisms. An important type of chromatin regulatory mechanism takes place on the tails of histone proteins in nucleosomes. These histone tail post-translational modifications (PTMs) are essential for normal cellular function and their misregulation has been shown to contribute to cancer cell formation. Histone tail PTMs are covalent in nature and, among others, include: phosphorylation, methylation, ubiquitylation, and acetylation. However, the one exception to the list of covalent histone tail modifications is cis-trans proline isomerization. In yeast, it has been shown that cis-trans proline isomerization on histone H3 proline 38 (H3-Pro38) can influence the methylation of a neighboring lysine residue. In addition, a yeast proline isomerase enzyme has been identified that can catalyze the interconversion of this proline isomerization event. This has led to the hypothesis that H3-Pro38 can act as a switch to control other histone modifications, recruit effector proteins, and alter the structure o chromatin. However, the ability to study proline isomerization has been severely limited by the lack of chemical tools that effectively mimic the cis and trans conformational states of proline. This proposed research will address this issue by developing H3-Pro38 peptide and protein mimics that are conformationally locked in either the cis or trans states. Such mimics do not currently exist and would be valuable in studying the effects of cis and trans proline on histone structure and function. For these studies, we will use chemical and peptide synthesis along with protein engineering techniques to develop proline peptide and protein mimics. With these chemical tools in hand, we will then use biochemical and biophysical approaches to explore the proposed biological roles of H3-Pro38 isomerization described above.
The specific aims of this research are: (1) To synthesize conformationally locked peptide mimics of histone H3-Pro38. (2) To characterize the role of H3-Pro38 isomerization on H3-Lys36 methylation/demethylation. (3) To identify human reader and writer proteins of cis and trans H3-Pro38. (4) To elucidate the structural role of cis and trans Pro38 isomerization on chromatin compaction. This proposed research is designed to provide much needed tools for studying H3-Pro38 cis-trans isomerization and, if successful, we expect this work to lay a foundation for understanding how proline switches regulate chromatin structure and function in humans.
Human DNA is packaged into cells in the form of chromatin which means that access to DNA for cellular processes must occur at the chromatin level, and that the regulation of chromatin has important consequences on physiological and pathophysiological cellular events including cancer cell formation. The goal of this proposed research is to investigate a poorly understood chromatin regulatory mechanism where proline amino acids act as a switch on histone protein tails to regulate chromatin structure and function. Efforts will focus on developing novel chemical tools that mimic different proline conformations and then using such tools to understand the effects of proline isomerization on chromatin regulation.
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