Testing the functional consequences of rapid centromeric DNA and protein evolution

测试着丝粒 DNA 和蛋白质快速进化的功能后果

• 批准号: 10785096
• 负责人: Piero Lamelza
• 金额: $ 10.34万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10785096
• 关键词: Affinity; Aneuploidy; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Process; Cell Line; Cells; Centromere; Chromatin Modeling; Chromosome Segregation; Chromosomes; Conflict (Psychology); Cytoplasm; DNA; DNA Binding; DNA Binding Domain; DNA Sequence; Data; Dicentric chromosome; Embryo; Embryonic Development; Eukaryota; Evolution; Exhibits; Female; Fertilization; Genetic; Genome; Goals; Histone H3; Hybrids; In Vitro; Infertility; Injections; Measures; Meiosis; Mitosis; Molecular Profiling; Mus; Nucleosomes; Organism; Orthologous Gene; Positioning Attribute; Process; Proteins; Role; Satellite DNA; Scaffolding Protein; Selfish DNA; Selfish Genes; Stretching; System; Techniques; Testing; Training; Untranslated RNA; Variant; career; centromere protein A; centromere protein C; chromatin remodeling; chromosome missegregation; egg; genome integrity; hybrid protein; mutant; novel; paralogous gene; preference; programs; protein function; reconstitution; recruit; reproductive; segregation; sperm cell; tool; zygote

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).

项目摘要/摘要 丝粒是一个蛋白质网络，该蛋白质通过含有A Cenp-A Paralog，CENP-A。尽管Centromere在染色体中扮演着保守的角色 隔离，其蛋白质的子集，对于其功能暴露的分子特征至关重要 自适应进化。重复的中心卫星DNA也迅速发展，序列差异很大 物种之间。 “ Centromere驱动假设”提案，即中心层面之间的遗传冲突 DNA和蛋白质会导致它们的快速进化。具体而言，CencerRomeric DNA自私地行事并增加 通过增加对丝粒蛋白的结合亲和力，它们通过女性减数分裂（驱动）遗传。自私 丝粒也对生物体有害，该生物可以选择具有A的新型丝粒蛋白质变体 对自私的DNA的亲和力降低，从而抑制了驱动器。 Centromere驱动假说进一步预测 在丝粒DNA-蛋白联合进化的独特爆发下，这种独特的进化谱系导致 在促进复制性分离的杂种中，有害的丝粒DNA-蛋白质不兼容。 我已经开发了一个新型的实验系统，以测试Centromere驱动器的三个关键建议 假设。首先是丝粒DNA重复变体不同募集的丝粒蛋白。经过 用不同的穆琳娜物种的精子施肥，肌肉菌的卵，我测试这两个物种是否 丝粒DNA的重复不同，它们从杂化合子细胞质中募集着丝粒蛋白的能力不同。 我的初步数据表明，pahari cenp-a核小体对钥匙具有更高的结合亲和力 丝粒蛋白支架CENP-C，比M.肌肉CENP-A核小体。为了我的第一个目标，我会 生物化学重构帕哈里菌Cenp-a核小体，并测试centromeric DNA是否 包裹意味着CENP-C结合的增加。为了我的第二个目标，我将测试是否快速发展 共粒蛋白直系同源物（变体）与中心粒DNA的结合不同。我会暂时表达各种各样的 杂化合子中的丝粒蛋白直系同源物，并确定其对两者的结合偏好是否 物种的中心粒不同。对于我的第三个目标，我将测试是否发散centromere dnas导致 杂种中有害不相容。我发现在M. Musculus / M. Pahari Hybrid zygotes中，M。 Pahari Centromeres拼写错误。我将生成更连续的M. Pahari基因组组件 确定该拼写错误的唯一帕哈里帕哈里中心DNA序列是否存在，并测试是否 错误地包装会导致有害的染色体错误分类。 我开发的混合胚胎系统是询问功能的强大工具 丝粒DNA和蛋白质的差异，但也通过解开自私的遗传冲突。 DNA来自其规格特定的补充剂。这个系统和我的培训将使我确定一个 围绕潜水员哺乳动物自私遗传元素的未来研究计划（请参阅职业目标）。