Evolution of Homologous recombination mechanisms

 描述(由申请人提供) 同源重组重组了亲本染色体之间的遗传信息，产生了遗传多样性并推动了进化。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决定的，PRDM9蛋白在组蛋白H3的赖氨酸4三甲基化的位置上，DNA双链断裂(DSB)的形成引发了重组。在Prdm9基因敲除小鼠中，DSB针对的是基因启动子和增强子，这些基因也带有H3K4三甲基化标记。因此，PRDM9引导重组远离功能基因组元件。这一作用是重要的，因为数据表明，重组在局部核苷酸水平和总染色体重排中都有突变效应。Prdm9基因敲除小鼠是不育的，配子体在同源重组诱导后不久就消失了。然而，一些哺乳动物缺乏典型的Prdm9基因。是什么定义了这类物种中的重组热点目前尚不清楚。为了深入了解可能的机制，我们建议研究两个非PRDM9生物的重组情况，并确定PRDM9的KRAB结构域在这些物种中可能表达的生物学功能。我们将使用我们的基于芯片/序列的方法，在(A)狗和(B)短尾负鼠中建立高分辨率的DSB热点基因组图谱。我们将把这些图谱与我们最近为小鼠和人类创建的图谱进行比较，以确定DSB热点的共同和不同特征及其在有PRDM9和缺乏PRDM9的动物中的分布。我们还将建立两个小鼠模型，以确定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，该品系仅限于没有DNA结合域的PRDM9的N-末端部分(在负鼠中发现的形式)和(B)表达具有突变的KRAB结构域的全长PRDM9的品系。我们将评估这两个模型的依赖于PRDM9的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种群的一般基因组组成方面的突出作用外，重组本身对于在配子发生过程中正确分离同源染色体是必不可少的。重组缺陷，包括重组效率降低和重组事件放置不当，总是与不孕不育、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，了解PRDM9的功能是极其重要的，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，以及哺乳动物物种形成的机制。

 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.