Evolution of Homologous recombination mechanisms

 描述（由申请人提供） 同源重组亲本染色体之间的遗传信息，产生遗传多样性和驱动进化。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，热点位置由PRDM 9蛋白的序列特异性结合限定，PRDM 9蛋白在随后通过形成DNA双链断裂（DSB）而引发重组的位点处使组蛋白H3的赖氨酸4三甲基化。在Prdm 9敲除小鼠中，DSB靶向基因启动子和增强子，其也携带H3 K4三甲基化标记。因此，PRDM 9引导重组远离功能基因组元件。这一作用很重要，因为数据表明在局部核苷酸水平和总染色体重排中重组的致突变作用。Prdm 9敲除小鼠是不育的，在诱导同源重组后不久配子体被消除。然而，一些哺乳动物缺乏典型的Prdm 9基因。目前尚不清楚这些物种中重组热点的定义。为了深入了解可能的机制，我们建议研究两种非PRDM 9生物体中的重组景观，并确定可能在这些物种中表达的PRDM 9的KRAB结构域的生物学功能。我们将采用我们的ChIP/seq为基础的方法来建立高分辨率的全基因组DSB热点图（a）的狗和（B）的短尾负鼠。我们将这些图谱与我们最近为小鼠和人类生成的图谱进行比较，以确定DSB热点的共同和不同特征及其在具有和缺乏PRDM 9的动物中的分布。我们还将生成两个小鼠模型以确定PRDM 9的KRAB结构域的生物学作用。这些将包括（a）表达限制于PRDM 9的N-末端部分而没有DNA结合结构域（在负鼠中发现的形式）的PRDM 9的截短形式的小鼠品系和（B）表达具有突变KRA B结构域的全长PRDM 9的品系。我们将评估两种模型的PRDM 9依赖性H3 K4 me 3标记的分布、启动同源重组的能力、DSB热点的分布和完成重组的能力。除了同源重组在建立群体的一般基因组组成中的突出作用之外，重组本身对于配子发生期间同源染色体的适当分离是必不可少的。重组缺陷，包括重组效率降低和重组事件的不适当放置，总是与不育、流产和非整倍体相关的出生缺陷相关。除了明显的健康影响外，从进化的角度来看，了解PRDM 9的功能非常重要，因为Prdm 9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重组效率和分布在哺乳动物中的机制，可能干扰重组进展的因素，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.