Defining the Minimal Trigger for Human Centromere Formation

人类着丝粒是由组蛋白H3变异体CENP-A指定的大染色体座位，通常在一系列α卫星重复序列中发现。由于着丝粒无处不在，而且是结构性遗传的，人们对它们最初的形成知之甚少。然而，通过见证着丝粒的诞生，我们可以发现着丝粒组件的设计原则。人类新着丝粒的形成在自然界中是一种罕见的事件，到目前为止，大多数研究都集中在α卫星DNA的异位染色体外阵列上或通过人工核化CENP-A染色质来播种着丝粒。虽然有洞察力，但这些方法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本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。最近，宿主实验室开发了一种CRISPR-Cas9驱动的着丝粒缺失技术，以分离自然形成的表观遗传种子的人类新着丝粒(Neo4p13)。我将利用这一方法来发现在完整的人类染色体上高效地进行新着丝粒播种所需的关键要素。具体地说，由于自发形成新着丝粒的情况很少见，我将确定两个关键着丝粒成分CENP-A核小体和α-DNA的最小拷贝数，它们足以有效地产生新着丝粒。此外，我还将比较活跃染色质和沉默染色质在这些新着丝粒成核中的作用。此外，我将通过在我们目前的新着丝粒附近和远端位置插入一个Alphid DNA阵列来确定Alphid DNA对现有新着丝粒的贡献。在基本着丝粒生物学和高级染色体工程的交界处，这一建议将提高我们在人类功能染色体的背景下可靠地建立稳定的着丝粒的能力。

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.