Understanding heterochromatin boundary architecture by dissecting the spatial regulation of the putative histone demethylase Epe1

通过剖析假定的组蛋白去甲基化酶 Epe1 的空间调控来了解异染色质边界结构

• 批准号: 529513809
• 负责人: Professor Dr. Sigurd Braun
• 金额: --
• 依托单位: Institut für Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/529513809?language=en
• 关键词: Understanding; heterochromatin; boundary; architecture; dissecting

This proposal aims to dissect the layers of regulation that control the local accumulation of a boundary factor on chromatin, ensuring its function as a barrier against the expansion of heterochromatin domains. Chromatin boundaries are crucial to separate ‘silent’ heterochromatin from ‘active’ euchromatin to ensure proper regulation of gene expression and cellular identity. While DNA elements have been identified that contribute to the recruitment of boundary factors, the spatial regulation of boundaries is highly complex. We previously demonstrated that the putative histone demethylase Epe1, which prevents ectopic spreading of heterochromatin in Schizosaccharomyces pombe (fission yeast), is spatiotemporally controlled on chromatin through ubiquitin-dependent degradation, confining Epe1 localization to the boundaries. This epigenetic concept of boundary formation differs from chromatin barriers established by DNA-binding sequences previously described. However, how Epe1 is protected from degradation at the boundaries and how it exerts its boundary function have remained elusive. Here, we seek to elucidate the mechanism of Epe1 regulation by dissecting the dynamic interactions with its multiple binding partners that control its recruitment, degradation, and protection. Our unpublished data indicate that removing the unstructured C-terminal region prevents Epe1 degradation, causing its accumulation on chromatin that triggers silencing defects at heterochromatin. We hypothesize that the C-terminal region, besides other functions, mediates Epe1 degradation but is masked at the boundaries. We seek to refine and separate these functions and identify the mechanisms protecting Epe1 from degradation. A candidate is the bromodomain protein Bdf2, which is recruited by Epe1 to the heterochromatin boundaries. We find that the loss of Bdf2 (bdf2∆) reduces the chromatin association of Epe1. Intriguingly, Epe1 levels can be restored in bdf2∆ cells when expressing truncated Epe1 that lacks the C-terminal part. Thus, we seek to test the hypothesis that Bdf2 protects Epe1 from ubiquitin-dependent degradation by masking its recognition or ubiquitylation sites. In addition, we will examine the dynamic interactions with Bdf2 and HP1 proteins, both contributing to Epe1 recruitment but at different chromatin regions. In particular, we seek to understand how differences in local Bdf2 and HP1 abundance drive Epe1’s ability to establish heterochromatin boundaries. The proposed work will shed light on the architecture of heterochromatin boundaries by providing mechanistic insights into how spatial regulation is coordinated through dynamic interactions across multiple protein partners.

该提案旨在剖析控制染色质上边界因子局部积累的调控层，确保其作为异染色质结构域扩展的屏障的功能。染色质边界对于将“沉默”异染色质与“活跃”常染色质分开以确保基因表达和细胞身份的适当调节至关重要。虽然DNA元件已被确定，有助于招募的边界因子，空间调控的边界是非常复杂的。我们以前证明，假定的组蛋白去甲基化酶Epe 1，防止异染色质在裂殖酵母（裂殖酵母）的异位扩散，是时空控制染色质通过泛素依赖性降解，限制Epe 1本地化的边界。这种边界形成的表观遗传学概念不同于先前描述的由DNA结合序列建立的染色质屏障。然而，如何保护Epe 1在边界处不被降解以及它如何发挥其边界功能仍然是难以捉摸的。在这里，我们试图阐明Epe 1调节的机制，通过解剖其多个结合伙伴，控制其招聘，降解和保护的动态相互作用。我们未发表的数据表明，去除非结构化的C-末端区域可以防止Epe 1降解，导致其在染色质上积累，从而引发异染色质的沉默缺陷。我们假设，C-末端区域，除了其他功能，介导Epe 1降解，但在边界被掩盖。我们试图完善和分离这些功能，并确定保护Epe 1免受降解的机制。一个候选者是布罗莫结构域蛋白Bdf 2，它被Epe 1募集到异染色质边界。我们发现Bdf 2（bdf 2 ️）的缺失减少了Epe 1的染色质结合。有趣的是，当表达缺少C-末端部分的截短的Epe 1时，Epe 1水平可以在bdf 2 cells中恢复。因此，我们试图测试的假设，Bdf 2保护Epe 1从泛素依赖性降解掩蔽其识别或泛素化位点。此外，我们将研究与Bdf 2和HP 1蛋白的动态相互作用，两者都有助于Epe 1的招募，但在不同的染色质区域。特别是，我们试图了解如何在当地的Bdf 2和HP 1丰度的差异驱动Epe 1的能力，建立异染色质边界。拟议的工作将阐明异染色质边界的架构，通过提供机制的见解，空间调控是如何通过跨多个蛋白质伙伴的动态相互作用进行协调。