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

这项建议旨在剖析控制染色质上边界因子局部积累的调控层，确保其作为阻止异染色质结构域扩张的屏障。染色质边界是区分“沉默的”异染色质和“活跃的”常染色质的关键，以确保基因表达和细胞特性的适当调节。虽然已经确定了有助于边界因子招募的DNA元件，但边界的空间调节是高度复杂的。我们先前已经证明，组蛋白去甲基酶Epe1可以阻止裂殖酵母(Schizosaccharmyces Pombe)中异染色质的异位扩散，它通过泛素依赖的降解在染色质上受到时空控制，将Epe1定位于边界。这种表观遗传的边界形成概念不同于由前面描述的DNA结合序列建立的染色质屏障。然而，Epe1如何在边界上保护免受退化，以及如何发挥其边界功能仍然难以捉摸。在这里，我们试图通过剖析Epe1与其多个结合伙伴的动态相互作用来阐明Epe1的调控机制，这些结合伙伴控制着Epe1的招募、降解和保护。我们未发表的数据表明，移除非结构化的C-末端区域可以防止Epe1的降解，导致其在染色质上积累，从而触发异染色质的沉默缺陷。我们假设，除了其他功能外，C-末端区域还参与Epe1的降解，但在边界处被屏蔽。我们试图提炼和分离这些功能，并确定保护Epe1免受降解的机制。一个候选是溴域蛋白Bdf2，它被Epe1招募到异染色质边界。我们发现BDF2(BDF2∆)的缺失降低了Epe1的染色质关联。有趣的是，当表达缺少C-末端部分的截短的Epe1时，BDF2∆细胞中的Epe1水平可以恢复。因此，我们试图验证Bdf2通过掩盖其识别或泛素化位点来保护Epe1免受泛素依赖的降解的假设。此外，我们还将研究与Bdf2和HP1蛋白的动态相互作用，这两种蛋白都有助于Epe1的招募，但位于不同的染色质区域。特别是，我们试图理解局部Bdf2和Hp1丰度的差异如何驱动Epe1的S建立异染色质边界的能力。这项拟议的工作将通过提供关于空间调控如何通过跨多个蛋白质伙伴的动态相互作用来协调的机械性见解，来阐明异染色质边界的架构。

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 Our unpublished data indicate that removing the unstructured C-terminal region prevents Epe1 degradation, causing its accumulation on chromatin that triggers silencing defects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.