The role of Spo11 in mammalian meiosis

Spo11 在哺乳动物减数分裂中的作用

• 批准号: 8553511
• 负责人: Rafael Camerini-Otero
• 金额: $ 52.41万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553511
• 关键词: Address; Affect; Apoptosis; Biological Process; Caenorhabditis elegans; Cancer Center; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Characteristics; Chemicals; Chromosome Pairing; Chromosome Segregation; Chromosomes; Cleaved cell; Collaborations; Complement; Cytoplasm; DNA; DNA Repair; DNA biosynthesis; DNA-PKcs; Data; Development; Double Strand Break Repair; Ensure; Eukaryota; Evolution; Exons; Female; Gene Expression Regulation; Genes; Genetic; Genetic Recombination; Genets; Genome; Goals; Homologous Gene; Human; Immunoglobulin Class Switching; Infertility; Knock-in Mouse; Knockout Mice; Life; Mammals; Meiosis; Meiotic Prophase I; Meiotic Recombination; Molecular Profiling; Monoclonal Antibodies; Mus; Nature; Oocytes; Organism; Oryctolagus cuniculus; Pathway interactions; Phenotype; Process; Prophase; Protein Isoforms; Proteins; RNA Splicing; Radiation; Role; SPO11 gene; Science; Shapes; Signaling Protein; Site; Somatic Cell; Spermatocytes; Spo11 protein; Sterility; Topoisomerase; Transgenes; Transgenic Mice; Wild Type Mouse; Work; ataxia telangiectasia mutated protein; embryonic stem cell; homologous recombination; insight; interest; male; man; prevent; programs

Summary of work: Chromosomal double-strand breaks (DSBs) pose a significant and immediate danger to genome integrity and cell survival. DSBs can arise through the action of exogenous agents (radiation and chemicals), endogenous causes (e.g., collapsed replication forks) and in the course of developmental programs (Ig recombination and class switching, and meiosis). DSBs are repaired by either nonhomologous end joining or homologous recombination (HR). Both pathways are equally important in most organisms, including mammals (reviewed in Sonoda et al. (2006) DNA Repair 5, 1021)). HR is common to all forms of life and is a multistep process involving many gene products. In eukaryotes, the biologically most important form of recombination is the exchange of genetic information between homologous chromosomes (homologs) in meiosis (reviewed in Gerton and Hawley (2005) Nature Rev. Genet. 6, 477). Meiotic recombination is the central phenomenon in the genetics of eukaryotes, ensures the proper segregation of chromosomes at the first division of meiosis (prevents non-disjunction) and is the main force shaping the evolution of genomes. In all organisms, homologous recombination is inextricably related to DNA repair and replication. Rad51 and the meiosis-specific Dmc1 protein, both homologues of bacterial RecA (the prototypical homologous recombination protein), have been identified in most eukaryotes including man and mouse, and homozygous Rad51 -/- mouse ES cells are not viable. Thus, the study of RecA homologues should yield insights into not only homologous recombination but also the regulation of gene stability and cell proliferation. In meiosis in all organisms, including mammals (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975), Spo11, a type II-like topoisomerase, cleaves the chromosomal DNA at many sites (a couple of hundred or more in mammals) in each and every nucleus (reviewed in Keeney and Neale (2006) Biochem. Soc. Trans. 34, 523). In mammals there are more endogenous DSBs in meiotic nuclei than in any somatic nuclei in the life of an organism, by a factor of at least 100. Hence the entire arsenal of the HR machinery invoked in somatic cells plus additional meiosis-specific proteins are required for the efficient repair of DSBs in meiosis. These include p53, Brca1, Brca2, Rad51, Dmc1, Atm, Atr, DNA-PKcs, Chk2, Nbsl, Mre11, Rpa, Blm, etc. Spo11 is required for meiotic chromosomal synapsis in S. cerevisae. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis (Romanienko and Camerini-Otero (2000) Mol. Cell 6, 975). Surprisingly, Spo11 heterozygosity rescues the meiotic prophase arrest seen in mice lacking the Atm (ataxia telangiectasia, mutated ) double-strand break signaling protein (Bellani, Romanienko, Cairatti and Camerini-Otero (2005) J. Cell Science 118, 3233). Most recently, we have used a rabbit monoclonal antibody to the Spo11 protein to confirm that in fact most of the Spo11 protein is expressed late in prophase I and is found in the cytoplasm. Furthermore, we have been able to show that it is the alpha-isoform that is most abudantly expressed and that is expressed late. Finally, we have reexamined the expression profiles of the two major splicing isoforms of Spo11, Spo11alpha (exon 2 skipped) and Spo11beta found in both mice and humans. Our data argues for a major role for Spo11beta in introducing the breaks that initiate meiotic recombination. Furthermore, we find that Spo11alpha is the form that is most expressed but mainly after the DSBs are introduced and this expression is seen in both spermatocytes and oocytes. We propose a role for Spo11alpha in mid- to late prophase, presumably acting as a topoisomerase, in both male and female meiocytes. In order to understand the role of SPO11 α, we have generated a transgenic mouse carrying a BAC with a modified Spo11 locus inserted ectopically into the genome. We were able to detect over-expression of SPO11 α in a wild type mouse carrying the transgene and found that over-expression of SPO11 α does not affect meiotic progression. We found that even though SPO11 α is expressed early in a Spo11-/- Spo11α +/- transgenic mouse, it fails to rescue the meiotic arrest characteristic of the Spo11 knockout mouse. This supports the notion that the SPO11 β isoform is responsible for introducing the breaks. At the moment we are trying to address whether SPO11α is able to rescue the KO phenotype after introducing DSBs exogenously. We plan to generate a knock-in mouse where the endogenous Spo11 locus would be replaced by the construction expressing only SPO11 α. Most recently, in a collaboration with the labs of Maria Jasin and Scott Keeney at Sloan Ketterin Cancer Center, we have been examining whether Spo11alpha can complement a sterile transgenic mouse carrying Spo11beta.

染色体双链断裂（DSB）对基因组完整性和细胞存活构成重大和直接的危险。 DSB可以通过外源性因素（辐射和化学品）、内源性原因（例如，折叠的复制叉）和发育程序过程中（IG重组和类别转换，以及减数分裂）。 DSB通过非同源末端连接或同源重组（HR）修复。这两种途径在大多数生物体中同样重要，包括哺乳动物（综述于Sonoda等人（2006）DNA Repair 5，1021））。 HR是所有生命形式所共有的，是一个涉及许多基因产物的多步骤过程。在真核生物中，生物学上最重要的重组形式是减数分裂中同源染色体（同系物）之间的遗传信息交换（综述于Gerton和Hawley（2005）Nature Rev. Genet. 6，477）。减数分裂重组是真核生物遗传学中的中心现象，确保了减数分裂第一次分裂时染色体的正确分离（防止不分离），并且是塑造基因组进化的主要力量。 在所有生物体中，同源重组与DNA修复和复制密不可分。Rad 51和减数分裂特异性Dmc 1蛋白，两者都是细菌RecA（原型同源重组蛋白）的同源物，已经在包括人和小鼠在内的大多数真核生物中鉴定，并且纯合Rad 51-/-小鼠ES细胞不能存活。 因此，RecA同源物的研究不仅要深入了解同源重组，而且要了解基因稳定性和细胞增殖的调控。 在包括哺乳动物在内的所有生物体的减数分裂中（Romanienko和Camerini-Obernik（2000）Mol. Cell 6，975），Spo 11，一种II型样拓扑异构酶，在每个细胞核中的许多位点（哺乳动物中有几百个或更多）切割染色体DNA（综述于Keeney和Neale（2006）Biochem.Soc.Trans.34，523）。在哺乳动物中，减数分裂核中的内源性DSB比生物体生命中的任何体细胞核中的内源性DSB多至少100倍。 因此，在体细胞中调用的HR机制的整个军火库加上额外的减数分裂特异性蛋白质是减数分裂中DSB的有效修复所必需的。 这些包括p53、Brca 1、Brca 2、Rad 51、Dmc 1、Atm、Atr、DNA-PKcs、Chk 2、Nbs 1、Mre 11、Rpa、Blm等。 Spo 11是S.啤酒。令人惊讶的是，Spo 11同源物在C. elegans和D.但仍是减数分裂重组所必需的。我们已经产生了一个SPO 11基因敲除小鼠，以研究该基因在哺乳动物中的生物学功能。小鼠SPO 11的破坏导致不育。精母细胞在粗线期之前停滞，几乎没有或没有同源突触，并经历细胞凋亡（Romanienko和Camerini-Obern（2000）Mol. Cell 6，975）。令人惊讶的是，Spo 11杂合性挽救了在缺乏Atm（共济失调毛细血管扩张症，突变的）双链断裂信号传导蛋白的小鼠中观察到的减数分裂前期停滞（Bellani，Romanienko，Cairatti和Camerini-Oscillation（2005）J. Cell Science 118，3233）。 最近，我们已经使用了兔单克隆抗体的Spo 11蛋白，以确认，事实上，大多数Spo 11蛋白的后期表达在前期I和细胞质中发现。 此外，我们已经能够表明，它是α-同种型，是最丰富的表达和表达晚。 最后，我们重新研究了在小鼠和人类中发现的Spo 11的两种主要剪接异构体Spo 11 α（外显子2跳过）和Spo 11 β的表达谱。 我们的数据认为Spo 11 β在引入启动减数分裂重组的断裂中起着重要作用。 此外，我们发现Spo 11 alpha是表达最多的形式，但主要是在引入DSB后，并且这种表达在精母细胞和卵母细胞中均可见。 我们提出了一个作用Spo 11 α中晚期前期，大概作为一个拓扑异构酶，在男性和女性的性母细胞。 为了了解SPO 11的作用，我们已经产生了一个转基因小鼠携带BAC与修改Spo 11位点异位插入到基因组中。我们能够在携带转基因的野生型小鼠中检测到SPO 11的过表达，并发现SPO 11的过表达不影响减数分裂进程。我们发现，即使SPO 11在Spo 11-/-Spo 11 +/-转基因小鼠中早期表达，但它未能挽救Spo 11敲除小鼠的减数分裂停滞特征。这支持了SPO 11亚型负责引入断裂的观点。目前，我们正试图解决SPO 11是否能够拯救KO表型后，引入DSB外源。我们计划产生敲入小鼠，其中内源性Spo 11基因座将被仅表达SPO 11的构建体取代。 最近，在与Sloan Ketterin癌症中心的Maria Jasin和Scott Keeney实验室的合作中，我们一直在研究Spo 11 alpha是否可以补充携带Spo 11 beta的不育转基因小鼠。