Assembling and recombining the Arabidopsis centromeres

当细胞分裂时，染色体必须自我复制并分离到相反的细胞两极。至关重要的是，每个子代细胞都要继承平衡数量的染色体。这一过程是通过染色体与纺锤体微管结合来实现的。染色体通过称为着丝粒的特殊区域附着在纺锤体上。没有正确地附着在纺锤体上可能会导致染色体分离的缺陷，并与癌症和不孕不育有关。着丝粒在确保染色体分离中的作用是一种古老的、在细胞中非常保守的功能。通常，当研究这种高度保守的过程时，所涉及的机制在不同物种之间非常相似。然而，令人惊讶的是，着丝粒的情况正好相反，与着丝粒相关的DNA序列和蛋白质是基因组中变化最快的。这种现象被称为“着丝粒悖论”。研究着丝粒的一个挑战是相关的DNA序列高度重复。例如，在许多物种中，着丝粒由短的(~170-180碱基对)序列组成，以头尾相连的方向多次复制(100-1000)。这些被称为卫星阵列，正是在这些序列中，微管将结合到染色体上。人们还知道着丝粒卫星阵列能够在物种之间迅速和广泛地变化，但这些卫星阵列是如何进化和变化的还知之甚少。非常高的重复度使得着丝粒基本上不可能用上一代短读DNA测序技术来研究。然而，随着包括牛津纳米孔在内的长读DNA测序技术的出现，新的机会正在出现。在这项提案中，我们将首次利用长阅读测序来组装模式植物拟南芥的着丝粒。我们将使用这些着丝粒的图谱来研究在胚系期间发生的重组过程如何有助于着丝粒的快速进化。这项工作的一个主要成果将是完成拟南芥基因组中的着丝粒缺口，我们将向社会公布这些缺口。除了特定的重复DNA序列外，着丝粒在细胞分裂期间需要表观遗传标记来发挥其功能。例如，一种名为CENH3的特殊组蛋白与着丝粒结合，对于染色体附着在纺锤体微管上至关重要。此外，着丝粒经常被DNA甲基化高度修饰，尽管这个表观遗传标记在着丝粒中的功能尚不清楚。因此，在最终目标中，我们将使用长阅读测序来研究缺乏DNA甲基化的拟南芥突变体中的着丝粒和重组。我们的工作将一起揭示着丝粒是如何构成的，以及它们是如何如此迅速地进化的。重要的是，在许多作物物种中，着丝粒周围的区域也受到重组的抑制，这可能会限制育种过程中的品系改良。因此，我们在该提案中产生的知识可能会提供解锁靠近着丝粒的重组的方法，以加速作物育种。

When cells divide the chromosomes must copy themselves and segregate to opposite cell poles. It is critical that each daughter cell inherits a balanced number of chromosomes. This process is achieved by the chromosomes binding to spindle microtubules. Chromosomes attach to the spindle via specialised regions called centromeres. A failure to attach to the spindle correctly can cause defects in chromosome segregation and is associated with cancer and infertility. The role of the centromeres in ensuring chromosome segregation is an ancient and deeply conserved function in cells. Typically, when such highly conserved processes are studied, the mechanisms involved are very similar between different species. However, surprisingly the opposite is true for the centromeres, and the DNA sequences and proteins associated with them are some of the fastest changing in the genome. This phenomenon is termed the 'centromere paradox'. One challenge to studying the centromeres is that the associated DNA sequences are highly repetitive. For example, in many species the centromeres consist of short (~170-180 base pairs) sequences copied many times (100s-1000s) in a tandem head-to-tail orientation. These are known as satellite arrays and it is within these sequences that the microtubules will bind to the chromosome. It is also known that the centromere satellite arrays are capable of rapid and extensive change between species, yet how these satellite arrays evolve and change is poorly understood. The very high degree of repetition has made the centromeres essentially impossible to study with the previous generation of short read DNA sequencing technologies. However, new opportunities are arising with the advent of long-read DNA sequencing technologies, including Oxford Nanopore. In this proposal we will harness long-read sequencing to assemble the centromeres of the model plant species Arabidopsis for the first time. We will use these maps of the centromeres to investigate how recombination processes occurring during the germline might contribute to the fast evolution of the centromeres. A major output from this work will be completion of the centromere gaps in the Arabidopsis genome, which we will release to the community.In addition to specific repeat DNA sequences, the centromeres are known to require epigenetic marks for their function during cell division. For example, a special histone protein called CENH3 binds to the centromeres and is critical for chromosomes to attach to the spindle microtubules. Additionally, centromeres are often highly modified by DNA methylation, although the function of this epigenetic mark in the centromeres is unknown. Therefore, in the final objective we will use long-read sequencing to investigate the centromeres and recombination in Arabidopsis mutants that lack DNA methylation. Together our work will reveal new insights into how the centromeres are structured and how they evolve so quickly. Importantly, in many crop species the regions surrounding the centromeres are also suppressed for recombination, which can limit strain improvement during breeding. The knowledge we generate in this proposal may therefore provide ways to unlock recombination close to the centromeres in order to accelerate crop breeding.