Evolution of Homologous recombination mechanisms

同源重组机制的演变

• 批准号: 9245713
• 负责人: Galina Petukhova
• 金额: $ 30.47万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-17 至 2020-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245713
• 关键词: Address; Affect; Alleles; Aneuploidy; Animals; Automobile Driving; Binding; Biological; Biological Process; Birth; Canis familiaris; ChIP-seq; Chromosomal Rearrangement; Chromosome Segregation; Chromosomes; Congenital Abnormality; DNA Binding Domain; DNA Double Strand Break; Data; Defect; Didelphidae; Early Diagnosis; Elements; Enhancers; Event; Evolution; Failure; Fertility; Gametogenesis; Genes; Genetic Recombination; Genetic Transcription; Genetic Variation; Genomic Segment; Genomics; Health; Heterozygote; Histone H3; Human; Hybrids; Infertility; Knockout Mice; Lead; Length; Link; Location; Lysine; Mammals; Maps; Measures; Meiosis; Modeling; Monodelphis; Monodelphis Domestica; Mus; N-terminal; Nucleotides; Organism; Peptide Initiation Factors; Population; Prevention; Process; Proteins; Pseudogenes; Resolution; Role; Site; Spontaneous abortion; Sterility; Vertebrates; base; functional genomics; genetic information; genetic variant; genome-wide; homologous recombination; insight; mouse model; mutant; promoter; public health relevance; segregation

 DESCRIPTION (provided by applicant) Homologous recombination reshuffles genetic information between parental chromosomes generating genetic diversity and driving evolution. Recombination predominantly occurs at a set of genomic locations called recombination hotspots. In most mammals, including human, hotspot locations are defined by the sequence specific binding of the PRDM9 protein, which tri-methylates lysine 4 of the histone H3 at the sites where recombination is later initiated by the formation of DNA double stranded breaks (DSBs). In Prdm9 knockout mice DSBs are targeted to gene promoters and enhancers, which also carry an H3K4 trimethylation mark. Therefore, PRDM9 directs recombination away from functional genomic elements. This role is important as data indicate mutagenic effects of recombination both at the local nucleotide level and in gross chromosomal rearrangements. Prdm9 knockout mice are sterile with gametocytes eliminated shortly after induction of homologous recombination. Nevertheless, some mammals lack a canonical Prdm9 gene. What defines recombination hotspots in such species is currently unknown. To gain essential insights into possible mechanisms we propose to investigate the recombination landscape in two non-PRDM9 organisms and to determine the biological function of the KRAB domain of PRDM9 that may be expressed in these species. We will employ our ChIP/seq-based approach to build high-resolution genome-wide maps of DSB hotspots in (a) the dog and (b) the short-tailed opossum. We will compare these maps to the maps we recently generated for mice and human to determine the common and different features of DSB hotspots and their distributions in animals that have and that lack PRDM9. We will also generate two mouse models to determine the biological role of the KRAB domain of PRDM9. These will include (a) a mouse line expressing the truncated version of PRDM9 restricted to the N-terminal part of PRDM9 without the DNA binding domain (the form that is found in opossums) and (b) the line expressing full length PRDM9 with a mutant KRAB domain. We will evaluate both models with respect to their distribution of PRDM9-dependent H3K4me3 marks, the ability to initiate homologous recombination, the distribution of DSB hotspots, and the ability to complete recombination. In addition to the prominent role of homologous recombination in establishing the general genomic makeup of the population, recombination per se is essential for proper segregation of homologous chromosomes during gametogenesis. Recombination defects, including reduced recombination efficiency and improper placement of recombination events, are invariably associated with infertility, miscarriage and aneuploidy-related birth defects. Beyond the obvious health implications understanding of PRDM9 function is extremely important from an evolutionary point of view as Prdm9 is the only known speciation gene in vertebrates. Our studies aim to unravel the mechanisms that affect recombination efficiency and distribution in mammals, the factors that may interfere with recombination progression, and the mechanisms involved in mammalian speciation.

 描述（由申请人提供） 同源重组重组亲本染色体之间的遗传信息，产生遗传多样性并驱动进化。重组主要发生在一组称为重组热点的基因组位置。在包括人类在内的大多数哺乳动物中，热点位置是由 PRDM9 蛋白的序列特异性结合来定义的，PRDM9 蛋白在随后通过形成 DNA 双链断裂 (DSB) 启动重组的位点处对组蛋白 H3 的赖氨酸 4 进行三甲基化。在 Prdm9 敲除小鼠中，DSB 靶向基因启动子和增强子，这些启动子和增强子也带有 H3K4 三甲基化标记。因此，PRDM9 引导重组远离功能基因组元件。这一作用很重要，因为数据表明重组在局部核苷酸水平和总体染色体重排中具有诱变作用。 Prdm9 基因敲除小鼠在诱导同源重组后不久就失去了配子细胞，导致不育。然而，一些哺乳动物缺乏典型的 Prdm9 基因。目前尚不清楚此类物种中重组热点的定义。为了获得对可能机制的重要见解，我们建议研究两种非 PRDM9 生物体中的重组景观，并确定可能在这些物种中表达的 PRDM9 KRAB 结构域的生物学功能。我们将采用基于 ChIP/seq 的方法来构建 (a) 狗和 (b) 短尾负鼠中 DSB 热点的高分辨率全基因组图谱。我们将这些图与我们最近为小鼠和人类生成的图进行比较，以确定 DSB 热点的共同和不同特征及其在具有和缺乏 PRDM9 的动物中的分布。我们还将生成两个小鼠模型来确定 PRDM9 的 KRAB 结构域的生物学作用。这些将包括 (a) 表达 PRDM9 截短版本的小鼠品系，其仅限于 PRDM9 N 端部分，不带 DNA 结合结构域（负鼠中发现的形式）和 (b) 表达具有突变 KRAB 结构域的全长 PRDM9 的品系。我们将评估这两个模型的 PRDM9 依赖性 H3K4me3 标记的分布、启动同源重组的能力、DSB 热点的分布以及完成重组的能力。除了同源重组在建立群体的一般基因组构成中的突出作用之外，重组本身对于配子发生过程中同源染色体的正确分离也是必不可少的。重组缺陷，包括重组效率降低和重组事件放置不当，总是与不孕、流产和非整倍体相关的出生缺陷有关。除了明显的健康影响外，从进化的角度来看，理解 PRDM9 的功能极其重要，因为 Prdm9 是脊椎动物中唯一已知的物种形成基因。我们的研究旨在揭示影响哺乳动物重组效率和分布的机制、可能干扰重组进程的因素以及参与哺乳动物物种形成的机制。