Testing the functional consequences of rapid centromeric DNA and protein evolution

项目摘要/摘要 着丝粒是由核小体扎根于着丝粒dna的蛋白质网络，含有一个 着丝粒特异性组蛋白H3 Paralog，CENP-A。尽管着丝粒在染色体中的保守作用 分离，其功能所必需的蛋白质的子集表现出快速的分子特征 适应性进化。重复的着丝粒卫星DNA也快速进化，在序列上急剧分化 在物种之间。着丝粒驱动假说提出着丝粒与着丝粒之间的遗传冲突 DNA和蛋白质导致了它们的快速进化。具体地说，着丝粒DNA的自私行为和增加 它们通过雌性减数分裂遗传(通过增加对着丝粒蛋白的结合亲和力来驱动)。自私 着丝粒对生物体也是有害的，生物体选择新的着丝粒蛋白变体与 降低了对自私DNA的亲和力，从而抑制了动力。着丝粒驱动假说进一步预测 不同的进化谱系经历了独特的着丝粒DNA-蛋白质共同进化，导致 促进生殖隔离的杂交中有害的着丝粒DNA-蛋白质不相容。 我开发了一个新的实验系统来测试着丝粒驱动的三个关键命题 假设。第一，着丝粒DNA重复变异体差异化地招募着丝粒蛋白。通过 用发散的小鼠亚科动物的精子使肌肉分枝杆菌的卵受精，我测试了这两个物种 着丝粒DNA重复序列在从杂交合子细胞质中招募着丝粒蛋白的能力上有所不同。 我的初步数据表明，帕哈里小鼠CENP-A核小体对密钥有较高的结合亲和力 着丝粒蛋白支架，CENP-C，而不是肌肉分支杆菌CENP-A核小体。为了我的第一个目标，我会 生化重建巴氏杆菌CENP-A核小体，并检测着丝粒DNA是否 包装它使CENP-C结合增加。对于我的第二个目标，我将测试快速进化 着丝粒蛋白同源异构体(变体)与着丝粒DNA有差异地结合。我将短暂地表达各种 着丝粒蛋白在杂交受精卵中的同源基因，并确定它们对两者的结合偏好 不同物种的着丝粒不同。为了我的第三个目标，我将测试不同的着丝粒DNA是否会导致 混合动力车中有害的不相容。我发现在肌肉分枝杆菌和帕哈里分枝杆菌的杂交受精卵中，分枝杆菌的一个子集。 帕哈里着丝粒包装错误。我将生成一个更连续的帕哈里分枝杆菌基因组组装 确定独特的帕哈里支原体着丝粒DNA序列是否存在这种错误包装，并测试 错误包装会导致有害的染色体错误分离。 我开发的杂交胚胎系统是一个强大的工具，不仅可以用来研究 着丝粒DNA和蛋白质的差异，但也有更广泛的遗传冲突，因为分离自私 来自特定物种抑制子的DNA。这个系统和我所受的训练将使我能够建立一种 未来围绕不同的哺乳动物自私基因元素的研究计划(见职业目标)。

PROJECT SUMMARY/ABSTRACT The centromere is a network of proteins rooted to centromeric DNA by nucleosomes containing a centromere-specific histone H3 paralog, CENP-A. Despite the centromere’s conserved role in chromosome segregation, a subset of its proteins that are essential for its function exhibit molecular signatures of rapid adaptive evolution. Repetitive centromeric satellite DNA also rapidly evolves, drastically diverging in sequence between species. The “centromere drive hypothesis” proposes that a genetic conflict between centromeric DNA and proteins causes their rapid evolution. Specifically, centromeric DNAs behave selfishly and increase their inheritance through female meiosis (drive) by increasing binding affinity for centromeric proteins. Selfish centromeres are also detrimental to the organism, which selects for novel centromeric protein variants with a lower affinity for the selfish DNA, thereby suppressing drive. The centromere drive hypothesis further predicts that distinct evolutionary lineages undergo unique bouts of centromeric DNA-protein co-evolution, leading to deleterious centromeric DNA-protein incompatibilities in hybrids that promote reproductive isolation. I have developed a novel experimental system to test three key propositions of the centromere drive hypothesis. The first is that centromeric DNA repeat variants differentially recruit centromere proteins. By fertilizing the eggs of M. musculus with the sperm of divergent Murinae species, I test whether the two species centromeric DNA repeats differ in their ability to recruit centromeric proteins from the hybrid zygote cytoplasm. My preliminary data indicate that Mus pahari CENP-A nucleosomes have a higher binding affinity for a key centromeric protein scaffold, CENP-C, than M. musculus CENP-A nucleosomes. For my first aim, I will biochemically reconstitute the M. pahari CENP-A nucleosome and test whether the centromeric DNA that wraps it imparts this increase in CENP-C binding. For my second aim, I will test whether rapidly evolving centromere protein orthologs (variants) differentially bind to centromere DNA. I will transiently express various centromere protein orthologs in hybrid zygotes and determine whether their binding preferences for the two species’ centromeres differ. For my third aim, I will test whether divergent centromere DNAs result in deleterious incompatibilities in hybrids. I found that in M. musculus / M. pahari hybrid zygotes, a subset of M. pahari centromeres are mispackaged. I will generate a more contiguous M. pahari genome assembly to determine if unique M. pahari centromeric DNA sequences underly this mispackaging, and test whether mispackaging causes deleterious chromosome missegregation. The hybrid embryo system that I developed serves as a powerful tool to interrogate not only functional divergence of centromeric DNA and proteins, but also genetic conflict more broadly by uncoupling selfish DNAs from their species-specific suppressors. This system and my training will position me to establish a future research program around diverse mammalian selfish genetic elements (see Career Goals).