PDF(Pubmed)
Abstract：
BACKGROUND: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics.
RESULTS: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins.
CONCLUSIONS: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology.
RESULTS: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins.
CONCLUSIONS: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology.
摘要：
背景：组蛋白泛素化修饰正在成为一系列生物学过程中涉及的关键表观遗传机制。泛素化核小体的体外重建对于阐明组蛋白泛素化对染色质动力学的影响至关重要。
结果：在这项研究中，我们引入了一种非变性组蛋白八聚体泛素化（NDHOU）方法，用于产生泛素或泛素样修饰的组蛋白八聚体。该方法需要共表达和纯化组蛋白八聚体，然后用1,3-二溴丙酮化学交联成泛素。我们证明了用这些八聚体重建的核小体显示出高度的同质性，使它们与体外生化测定高度兼容。这些泛素化核小体在功能和结构上模拟生理底物。此外,我们已经将这种方法扩展到交联各种组蛋白八聚体和三种泛素样蛋白。
结论：总体而言，我们的发现为产生泛素化核小体提供了一种有效的策略，推进染色质生物学领域的生化和生物物理研究。
结果：在这项研究中，我们引入了一种非变性组蛋白八聚体泛素化（NDHOU）方法，用于产生泛素或泛素样修饰的组蛋白八聚体。该方法需要共表达和纯化组蛋白八聚体，然后用1,3-二溴丙酮化学交联成泛素。我们证明了用这些八聚体重建的核小体显示出高度的同质性，使它们与体外生化测定高度兼容。这些泛素化核小体在功能和结构上模拟生理底物。此外,我们已经将这种方法扩展到交联各种组蛋白八聚体和三种泛素样蛋白。
结论：总体而言，我们的发现为产生泛素化核小体提供了一种有效的策略，推进染色质生物学领域的生化和生物物理研究。
