Human THAP9-an active DNA transposase

描述（由申请人提供）： 人类基因组由> 50%的转座因子组成，转座因子是人类遗传变异的重要来源。3%的人类基因组由基于DNA的（剪切-粘贴）转座子组成，并且与转座因子相关的47个基因中的43个源自这些移动的因子。除了淋巴特异性RAG 1/RAG 2 V（D）重组酶和SETMAR蛋白（mariner转座酶和组蛋白甲基化酶SET结构域之间的融合体）外，这些基因中的大多数仍未被表征。这个转座子相关基因集合中的一个成员被称为THAP 9，它沿着其整个长度与果蝇P元件转座酶蛋白同源。THAP 9和其他11个人类基因含有THAP结构域，这是一个新识别的C2CH锌配位位点特异性DNA结合结构域。在真核生物的基因组中存在> 300个含有THAP结构域的蛋白质，但只有THAP 9在THAP结构域之外是同源的。比较基因组分析表明，THAP 9基因存在于21个脊椎动物基因组调查，但没有从啮齿动物和有P元件的末端反向重复序列在斑马鱼基因组中的转座子。这些观察结果表明THAP 9基因来源于古老的转座子，非常像V（D）J重组-RAG 1/RAG 2重组酶系统的进化情况。我们希望表征人类THAP 9基因，我们的初步数据表明它是一个活跃的DNA转座酶。这项研究的长期目标是了解转座因子相关基因在人类基因组中的功能。因为转座因子引起突变、基因组重排、导致遗传变异和改变基因表达，所以它们是基因组进化和基因表达程序的重要组成部分。本提案中概述的实验将表征THAP 9蛋白的功能活性，并可能鉴定出一个新的人类转座因子家族。该提议的中心假设是人类THAP 9基因编码活性DNA转座酶。我们将以几种方式测试这一假设，并试图在人类基因组中找到使用THAP 9蛋白作为转座酶的活性转座因子。这一提议背后的基本原理是在人类中发现一种活跃的基于DNA的转座子系统。为了解决THAP 9在人类细胞中发挥的功能作用，我们将：1）研究和表征人类THAP 9在细胞中的活性; 2）使用染色质免疫沉淀-HTP测序（ChIP-seq）绘制人类THAP 9基因组DNA结合位点; 3）人类THAP 9蛋白的蛋白质组学和生物化学分析。

DESCRIPTION (provided by applicant): The human genome is composed of > 50 % transposable elements, which are an important source of human genetic variation. 3% of the human genome consists of DNA-based (cut-and-paste) transposons and 43 of the 47 genes related to transposable elements are derived from these mobile elements. Most of these genes remain uncharacterized with the exception of the lymphoid-specific RAG1/RAG2 V(D) recombinase and the SETMAR protein, a fusion between a mariner transposase and a histone methylase SET domain. One member of this collection of transposon-related genes is called THAP9, which is homologous along its entire length to the Drosophila P element transposase protein. THAP9 and eleven other human genes contain a THAP domain, a newly recognized C2CH zinc-coordinating site-specific DNA binding domain. There are > 300 THAP domain-containing proteins in the genomes of eukaryotes, but only THAP9 is homologous beyond the THAP domain. Comparative genomic analyses indicate that the THAP9 gene is present in 21 vertebrate genomes surveyed, but is absent from rodents and that there are P element-like transposons with terminal inverted repeats in the zebrafish genome. These observations suggest that the THAP9 genes were derived from ancient transposons, much like the scenario for evolution of the V(D)J recombination- RAG1/RAG2 recombinase system. We wish to characterize the human THAP9 gene, which our preliminary data indicate is an active DNA transposase. The long-term goal of this research is to understand how genes related to transposable elements function in the human genome. Because transposable elements cause mutations, genome rearrangements, lead to genetic variation and alter gene expression, they are important components of genome evolution and gene expression programs. The experiments outlined in this proposal will characterize the functional activities of the THAP9 protein, and will potentially identify a new family of human transposable elements. The central hypothesis of this proposal is that the human THAP9 gene encodes an active DNA transposase. We will test this hypothesis in several ways and attempt to find active transposable elements in the human genome that use the THAP9 protein as a transposase. The rationale behind this proposal is to discover an active DNA-based transposon system in humans. In order to address the functional role THAP9 plays in human cells, we will: 1) Investigate and characterize the activities of human THAP9 in cells; 2) Map human THAP9 genomic DNA binding sites using chromatin immunoprecipitation-HTP sequencing (ChIP-seq); 3) Proteomic and biochemical analysis of human THAP9 protein.