组蛋白琥珀酰化对核小体和染色质动态结构影响的表征及其阅读器的鉴定

In eukaryotic cells, chromatin is a highly dynamic structure involved in the regulation of various cellular processes requiring access to DNA, including gene transcription, DNA replication, and DNA damage repair. The nucleosome is the fundamental repeating unit of chromatin, in which DNA tightly wraps around an octameric complex of histone proteins. Increasing evidence has suggested that posttranslational modifications (PTMs) of histones play important roles in the regulation of nucleosome and chromatin dynamics. In the meantime, histone PTMs may recruit effector proteins (readers) that specifically recognize the modifications to the chromatin to carry out downstream cellular events. Given numerous links between histone PTMs and normal cell physiology, abnormalities in their regulation may perturb chromatin structure, causing genomic instability and leading to human diseases such as cancer. Identification and characterization of histone PTMs are critical to fully understanding the regulatory mechanisms of different cellular processes. Although different types of histone PTMs have been reported, only a handful of them have been studied extensively. The biological functions and regulatory mechanisms of many histone PTMs, particularly newly identified PTMs, remain largely unknown. Characterization of these novel PTMs is, therefore, essential to decipher their biological significance. .The proposed research will focus on the characterization of a novel PTM on histones, lysine succinylation (Ksucc), which is a covalent modification at the epsilon-amino group of the lysine side chain by a succinyl group. Considering the fact that succinyl is a bulky group carrying a negatively charged carboxylate, histone succ u is likely to affect chromatin structure and dynamics. In turn, it may affect chromatin-templated processes. Meanwhile, histone Ksucc is also likely to recruit specific readers to the chromatin to signal downstream cellular processes. In this project, we will use chemical approaches in combination with biochemical, biophysical and cell biological approaches to examine the mechanisms and functions of histone Ksucc in the regulation of nucleosome and chromatin structure and dynamics. We will also carried out comprehensive chemical proteomics profiling for the identification of Ksucc readers. The completion of this project will help us understand the roles of histone Ksucc in the chromatin biology. In the long term, the findings from this project will lay the foundation for elucidating how this novel histone modification may contribute to human health and disease.

组蛋白翻译后修饰通过直接影响核小体和染色质动态结构，或者通过招募特异性阅读器作用于染色质，参与众多需要DNA可及性细胞过程的调控。目前已知的多种组蛋白修饰中仅有极少数被深入研究。而对于大多数，尤其是新发现的修饰，国内外对其生物学意义和功能的研究鲜有报道。此项目的研究将着重于一种新发现的组蛋白修饰，赖氨酸琥珀酰化。琥珀酰化不仅抹除了正常生理条件下赖氨酸残基的正电荷，更引入一个带有负电荷的羧酸片段。在染色质中，这一电性反转有可能显著改变核小体和染色质的结构, 从而影响基于染色质的生物学过程。同时，琥珀酰化也可能招募未知的特异性阅读器来发挥其生物学功能。我们将综合运用多学科手段，阐释组蛋白琥珀酰化影响核小体和染色质动态结构的分子机制；在全细胞蛋白质组水平,系统全面地鉴定及表征组蛋白琥珀酰化的阅读器。完成此研究有助于解析组蛋白琥珀酰化如何在复杂的“组蛋白密码”调控网络中发挥其生物学作用。

组蛋白的翻译后修饰（posttranslational modifications, PTMs）参与基因表达调控并在其中起到重要作用。组蛋白翻译后修饰通过直接影响核小体和染色质动态结构，或者通过招募特异性阅读器作用于染色质，参与众多细胞调控。目前已知的多种组蛋白修饰中仅有极少数被深入研究。而对于大多数，尤其是新发现的修饰，国内外对其生物学意义和功能的研究鲜有报道。此项研究着重于两种新发现的组蛋白修饰，赖氨酸琥珀酰化修饰（Ksucc）和戊二酰化修饰（Kglu）。该修饰不仅抹除了正常生理条件下赖氨酸残基的正电荷，更引入一个带有负电荷的羧酸片段。在染色质中，这一电性反转有可能显著改变核小体和染色质的结构, 从而影响基于染色质的生物学过程。同时，琥珀酰化和戊二酰化修饰也可能招募未知的特异性阅读器来发挥其生物学功能。因此，研究赖氨酸琥珀酰化和戊二酰化对于核小体动态稳定结构的影响以及系统全面的鉴定该修饰的调控和识别蛋白，对于理解该修饰的生物学意义以及功能尤为重要。.在本项目中，通过化学合成手段，我们得到了带有单一位点Ksucc/glu修饰的一系列组蛋白，包括：H2BK34位点琥珀酰化修饰的组蛋白类似物H2BKc34succ，H4K77位点琥珀酰化修饰的组蛋白H4K77succ,以及H4K91位点戊二酰化修饰的组蛋白H4K91glu。.并通过一系列体外表征手段，证明了Ksucc/glu修饰会破坏核小体内部组蛋白-组蛋白或组蛋白-DNA之间的离子键，从而降低核小体上的稳定性。利用芽殖酵母，我们发现同位点的、用以模拟负电荷修饰的谷氨酸突变(K-to-E)会影响染色质结构，从而激活细胞转录水平，并同时阻碍细胞周期、DNA损伤修复等过程。该研究项目的成果在于：1）首次阐明琥珀酰化修饰和戊二酰化修饰对于调控核小体、染色质稳定性的分子机理；2）系统、全面的阐明了该修饰对于与DNA相关的生理活动的调控机理；3）鉴定了H4K91glu的‘擦除器’和‘书写器’，并验证了该调控蛋白在与相关的生理活动中的功能与意义。