Defining the Minimal Trigger for Human Centromere Formation

定义人类着丝粒形成的最小触发因素

• 批准号: EP/X025675/1
• 负责人: Lars Jansen
• 金额: $ 24.26万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FX025675%2F1
• 关键词: Defining; Minimal; Trigger; Human; Centromere

Human centromeres are large chromosomal loci specified by the presence of the histone H3 variant CENP-A, typically found at an array of alpha-satellite repeat sequences. As centromeres are omnipresent and constitutively inherited, little is known about their initial formation. Yet, witnessing the birth of a centromere allows us to discover the design principles of a centromere assembly. Human neocentromere formation is a rare event in nature and most studies to date have focused on seeding centromeres either on ectopic extrachromosomal arrays of alpha-satellite DNA or by nucleating CENP-A chromatin artificially. While insightful, these approaches do not inform us about the underlying chromatin and genomic characteristics required for neocentromere specification. Yet, understanding how to build a centromere is key to basic chromosome biology and will instruct the engineering of synthetic mammalian chromosomes.Recently, the host laboratory developed a CRISPR-Cas9-driven centromere deletion technology to isolate a spontaneously-formed epigenetically seeded human neocentromere (Neo4p13). I will capitalize on this methodology to discover what are the key elements needed for efficient neocentromere seeding on an intact human chromosome. Specifically, as spontaneous neocentromere formation is rare, I will determine the minimal copy number of two key centromere ingredients, CENP-A nucleosomes and alphoid DNA, that is sufficient to efficiently generate neocentromeres. Moreover, I will compare the role of active and silenced chromatin in the nucleation of these neocentromeres. Further, I will define the contribution of alphoid DNA to existing neocentromeres through the insertion of an alphoid DNA array adjacent to our current neocentromere, and at a distal site. At the interface of fundamental centromere biology and advanced chromosome engineering, this proposal will improve our ability to reliably build stable centromeres in the context of functional human chromosomes.

人类着丝粒是由组蛋白H3变体CENP-A的存在所指定的大型染色体基因座，通常发现于一系列α卫星重复序列处。由于着丝粒是普遍存在和组成性遗传，很少有人知道他们的最初形成。然而，见证了一个着丝粒的诞生，让我们发现了一个着丝粒组装的设计原则。人类新着丝粒的形成在自然界中是一个罕见的事件，迄今为止，大多数研究都集中在将着丝粒接种在α-卫星DNA的异位染色体外阵列上或通过人工使CENP-A染色质成核来接种着丝粒。虽然这些方法很有见地，但并没有告诉我们新着丝粒规范所需的潜在染色质和基因组特征。然而，了解如何构建着丝粒是基础染色体生物学的关键，并将指导合成哺乳动物染色体的工程化。最近，宿主实验室开发了一种CRISPR-Cas9驱动的着丝粒缺失技术，以分离自发形成的表观遗传种子人类新着丝粒（Neo 4p 13）。我将利用这种方法来发现在完整的人类染色体上有效接种新着丝粒所需的关键要素。具体来说，由于自发的新着丝粒形成是罕见的，我将确定两个关键的着丝粒成分，CENP-A核小体和α DNA，这是足以有效地产生新着丝粒的最小拷贝数。此外，我将比较活跃和沉默的染色质在这些neocentromeres成核的作用。此外，我将通过在我们目前的新着丝粒附近和远端插入α DNA阵列来定义α DNA对现有新着丝粒的贡献。在基本的着丝粒生物学和先进的染色体工程的界面上，该建议将提高我们在功能性人类染色体的背景下可靠地构建稳定的着丝粒的能力。