The biogenesis of nucleosome arrays and their decline during cellular aging

核小体和核小体重塑酶对人类健康至关重要。例如，衰老会破坏核小体的景观，破坏基因组的稳定，核小体重塑体的突变是癌症的驱动因素。基因组的大部分组装成核小体。大多数核小体被认为像串珠一样排列在DNA上，形成间隔均匀的核小体阵列。ISWI， Chd1和-正如我们最近提出的- INO80核小体重塑酶似乎催化均匀间距。核小体之间的间隔很难测量，因为大多数检测，包括MNase-seq，必须破坏阵列来检测核小体，所以只能提供间隔均匀的间接证据。大多数技术也不能检测DNA在某个基因组位置被核小体结合的部分，称为核小体占用。我们和其他人已经开发了长读核小体作图技术，因为它们为这些问题提供了解决方案。它们可以检测核小体的占用，并有可能揭示单个DNA分子上核小体之间的间隔。我们使用酶将核小体之间的DNA甲基化。然后，长读测序直接检测甲基化和非甲基化碱基的位置，从而揭示核小体的足迹。这项足迹技术将在整个提案中发挥重要作用。在Aim 1中，我们利用我们的工程酵母菌株，可以清晰地分离出INO80的活性。通过甲基化足迹，我们将直接证实INO80在体内均匀间隔核小体的假设。然后，我们将对INO80在体内的重塑活性进行结构-功能分析。目的2剖析转录，我们发现这一过程对核小体阵列具有深刻的破坏性。我们将严格测试我们的模型，即转录破坏核小体之间的均匀间隔，并评估十年来的假设，即转录部分或甚至完全驱逐核小体。我们通过快速耗用聚合酶II和可视化在转录组快速、应激诱导重编程过程中发生的核小体景观变化来做到这一点。在Aim 3中，我们揭示了细胞如何应对剧烈的核小体损失，这被认为发生在衰老过程中。我们将直接可视化在衰老过程中核小体景观的变化，包括在酵母和人类原代细胞中伴随的核小体损失。我们还验证了我们的假设，即核小体重塑者充当酵母基因组的守护者，防止过早衰老，并在衰老的人类细胞恢复活力时跟踪核小体景观的变化。通过对比酵母和人类的结果，我们将得出真核生物衰老的保守原则。我们对产生和破坏核小体阵列的酶的解剖可以作为未来研究相关酶的蓝图。研究结果有可能更好地理解和治疗与年龄有关的症状和疾病。

Nucleosomes and nucleosome remodeling enzymes are crucial to human health. Aging, for instance, disrupts the nucleosome landscape, destabilizing the genome, and mutations in nucleosome remodelers are drivers of cancers. Large fractions of the genome assemble into nucleosomes. Most nucleosomes are thought to arrange on DNA like beads on a string, forming arrays of evenly spaced nucleosomes. ISWI, Chd1 and -as we recently proposed- INO80 nucleosome remodeling enzymes appear to catalyze even spacing.Even spacing between nucleosomes is surprisingly difficult to measure because most assays, incl. MNase-seq, must destroy arrays to detect nucleosomes, so that only indirect evidence for even spacing is provided. Most techniques also cannot detect the fraction of DNA at a certain genomic location that is bound by a nucleosome, termed the nucleosome occupancy. We and others have developed long-read nucleosome mapping techniques as they offer a solution to these problems. They natively detect nucleosome occupancy and carry the potential to reveal even spacing between nucleosomes on individual DNA molecules. We employ enzymes that methylate the DNA in between nucleosomes. Long-read sequencing then directly detects the positions of methylated and unmethylated bases, and thereby reveals the footprints of nucleosomes.This footprinting technology will be instrumental throughout this proposal. In Aim 1, we exploit our engineered yeast strain, in which INO80’s activity can be cleanly isolated. By methylation footprinting, we will directly confirm our hypothesis that INO80 evenly spaces nucleosomes in vivo. We will then engage in a structure-function analysis of INO80’s remodeling activity in vivo.Aim 2 dissects transcription, a process we find to be deeply disruptive to nucleosome arrays. We will critically test our model that transcription destroys even spacing between nucleosomes, and assess decade-old hypotheses that transcription partially or even fully evicts nucleosomes. We do so by rapid depletion of Polymerase II and by visualizing the changes to the nucleosome landscape that take place during rapid, stress-induced reprogramming of the transcriptome.In Aim 3 we reveal how cells cope with drastic nucleosome loss, which is proposed to occur during aging. We will directly visualize changes to the nucleosome landscape during aging, including concomitant nucleosome loss, in both yeast and human primary cells. We also test our hypothesis that nucleosome remodelers act as guardians of the yeast genome against premature aging, and follow changes to the nucleosome landscape upon rejuvenation of aged human cells. By contrasting results from yeast and human, we will derive conserved principles of aging across eukaryotes.Our dissections of enzymes generating and destroying nucleosome arrays may serve as a blueprint for studying related enzymes in the future. The results carry the potential to better understand and treat age-related symptoms and diseases.