Human THAP9-an active DNA transposase

描述(由申请人提供)： 人类基因组由50%的转座元件组成，这是人类遗传变异的重要来源。人类基因组的3%由基于DNA的(剪切和粘贴)转座子组成，与转座元件相关的47个基因中有43个来自这些可移动元件。除了淋巴特异的RAG1/RAG2 V(D)重组酶和SETMAR蛋白外，这些基因中的大多数仍然没有特征，SETMAR蛋白是水手转座酶和组蛋白甲基化酶SET结构域的融合。这个转座子相关基因集合中的一个成员被称为THAP9，它在整个长度上与果蝇P元素转座酶蛋白同源。THAP9和其他11个人类基因包含一个TAP结构域，这是一个新发现的C2CH锌协调位点特异性DNA结合域。真核生物基因组中存在含有&GT；300TAP结构域的蛋白质，但只有THAP9在TAP结构域之外是同源的。比较基因组学分析表明，THAP9基因存在于所调查的21个脊椎动物基因组中，但在啮齿动物中缺失，斑马鱼基因组中存在末端反向重复的P元件转座子。这些观察结果表明，THAP9基因来自古老的转座子，与V(D)J重组-RAG1/RAG2重组酶系统的进化非常相似。我们希望鉴定人类的THAP9基因，我们的初步数据表明它是一种活性的DNA转座酶。这项研究的长期目标是了解与转座元件相关的基因在人类基因组中的功能。由于转座元件引起突变、基因组重排、导致遗传变异和改变基因表达，它们是基因组进化和基因表达程序的重要组成部分。这项提案中概述的实验将表征THAP9蛋白的功能活性，并可能识别一类新的人类转座元件。这一提议的中心假设是，人类THAP9基因编码一种活跃的DNA转座酶。我们将通过几种方法验证这一假设，并试图在人类基因组中找到使用THAP9蛋白作为转座酶的活性转座元件。这项提议背后的理论基础是在人类中发现一种活跃的基于DNA的转座子系统。为了探讨THAP9在人类细胞中所起的功能作用，我们将：1)研究和鉴定人THAP9在细胞中的活性；2)使用染色质免疫沉淀-HTP测序(CHIP-SEQ)定位人THAP9基因组DNA结合位点；3)对人THAP9蛋白进行蛋白质组学和生化分析。

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.