Analysis of Proline Isomerization in Epigenetics

描述(申请人提供)：本培训计划中建议的研究将集中于研究顺-反式脯氨酸异构化对染色质结构和功能的影响。真核细胞中DNA的生理状态是以染色质的形式出现的。因此，获得基本细胞过程的基因组，如细胞分裂、分化和动态平衡，必须发生在染色质水平。这导致了对染色质调节机制的广泛研究。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。这些组蛋白尾部翻译后修饰(PTM)对于正常的细胞功能是必不可少的，它们的错误调控已被证明有助于癌细胞的形成。组蛋白尾部PTM本质上是共价的，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的一个例外是顺式-反式脯氨酸异构化。在酵母中，已经证明组蛋白H3-Pro38(H3-Pro38)上的顺-反式脯氨酸异构化可以影响邻近赖氨酸残基的甲基化。此外，还鉴定了一种酵母脯氨酸异构酶，它可以催化这一脯氨酸异构化事件的相互转化。这导致了一种假设，即H3-Pro38可以作为开关来控制其他组蛋白修饰，招募效应蛋白，并改变染色质的结构。然而，由于缺乏有效模拟脯氨酸顺式和反式构象的化学工具，研究脯氨酸异构化的能力受到严重限制。这项拟议的研究将通过开发构象锁定在顺式或反式状态的H3-Pro38多肽和蛋白质模拟物来解决这一问题。这种模拟目前还不存在，在研究顺式和反式脯氨酸对组蛋白结构和功能的影响方面将是有价值的。对于这些研究，我们将使用化学和多肽合成以及蛋白质工程技术来开发脯氨酸多肽和蛋白质模拟物。有了这些化学工具，我们将使用生化和生物物理方法来探索上述H3-Pro38异构化的拟议生物学作用。本研究的具体目的是：(1)合成组蛋白H3-Pro38的构象锁定多肽模拟物。(2)研究H3-Pro38异构化在H3-Lys36甲基化/去甲基化中的作用。(3)鉴定人类顺式和反式H3-Pro38的读写器蛋白。(4)阐明顺式和反式Pro38异构化在染色质紧凑中的结构作用。这项拟议的研究旨在为研究H3-Pro38顺反异构化提供迫切需要的工具，如果成功，我们希望这项工作将为理解Pro开关如何调节人类染色质的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.