Analysis of Proline Isomerization in Epigenetics

项目描述（申请人提供）：本培训计划中拟开展的研究将侧重于研究顺反脯氨酸异构化对染色质结构和功能的影响。真核细胞中DNA的生理状态以染色质的形式存在。因此，进入基因组的基本细胞过程，如细胞分裂，分化和稳态必须发生在染色质水平。这导致了对染色质调控机制的广泛研究。一种重要的染色质调节机制发生在核小体中组蛋白的尾部。这些组蛋白尾翻译后修饰（PTM）对于正常细胞功能是必需的，并且它们的失调已被证明有助于癌细胞形成。组蛋白尾部PTM在性质上是共价的，并且其中包括：磷酸化、甲基化、泛素化和乙酰化。然而，共价组蛋白尾部修饰列表的一个例外是顺-反脯氨酸异构化。在酵母中，已经表明组蛋白H3脯氨酸38（H3-Pro38）上的顺-反脯氨酸异构化可以影响相邻赖氨酸残基的甲基化。此外，已经鉴定了可以催化这种脯氨酸异构化事件的相互转化的酵母脯氨酸异构酶。这导致了H3-Pro38可以作为控制其他组蛋白修饰、募集效应蛋白和改变染色质结构的开关的假设。然而，研究脯氨酸异构化的能力受到严重限制，缺乏有效地模拟脯氨酸的顺式和反式构象状态的化学工具。这项拟议的研究将通过开发在顺式或反式状态下构象锁定的H3-Pro38肽和蛋白质模拟物来解决这个问题。这种模拟物目前还不存在，将是有价值的研究顺式和反式脯氨酸对组蛋白的结构和功能的影响。对于这些研究，我们将使用化学和肽合成沿着与蛋白质工程技术开发脯氨酸肽和蛋白质模拟物。有了这些化学工具，我们将使用生物化学和生物物理方法来探索上述H3-Pro38异构化的生物学作用。本研究的具体目的是：（1）合成组蛋白H3-Pro38构象锁定肽模拟物。(2)表征H3-Pro38异构化对H3-Lys 36甲基化/去甲基化的作用。(3)鉴定人类顺式和反式H3-Pro38的读写蛋白。(4)阐明顺式和反式Pro38异构化对染色质致密化的结构作用。这项拟议的研究旨在为研究H3-Pro38顺反异构化提供急需的工具，如果成功，我们希望这项工作为了解脯氨酸开关如何调节人类染色质结构和功能奠定基础。

DESCRIPTION (provided by applicant): 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.