Interactions of MuLV IN with host proteins and DNA

 描述(由申请人提供)：小鼠白血病病毒优先整合在活性启动子区域、转录起始点(TSS)和CpG岛附近。整合的位置与病毒的致癌潜力有关，因此它的致病机制和用作基因传递载体。我们最近的合作研究发现，宿主BET家族蛋白，特别是Brd 2、3和4蛋白是MLV IN的相互作用因子，这种相互作用影响到TSS和CpG岛的整合。这为我们理解MLV靶点选择的机制提供了第一个窗口。这项应用研究了MLV IN和BET家族成员之间新定义的病毒-宿主相互作用。四个具体目标建立在两个关键观察结果的基础上。第一个是将Brd ET结构域与MLV IN C-末端结构域的结合联系起来。第二个结果表明，缺少C-末端23个氨基酸的IN蛋白不再偏向TSS和CpG岛的整合。在本实验室MLV IN CTD溶液结构的基础上，第一个具体目标是确定MLV IN CTD：Brd3 Et络合物的核磁共振结构。此外，还将对CTD与交叉DNA整合中间体形成的复合体进行结构分析。第二个特定目的是应用突变和生化分析来确定主要的和潜在的次要的Brd结合位点。在结合BET蛋白中，IN CCDs的争议性作用将被研究。MLV IN蛋白与已知ET结构域相互作用的能力将被研究。第三个具体目的是利用下一代测序和生物信息学来探测一组IN蛋白质的靶位偏好，无论是在整合部位的局部还是在人类基因组内的全局。检测了核小体结构中靶DNA的识别。最终的具体目标是将这些信息应用于开发有针对性的整合载体。这项研究将我们对MLV IN的深入知识转化为快速了解这种与宿主靶细胞的相互作用。这项研究解决了与靶向MLV整合到启动子区域的机制有关的突出问题之一。这些研究有可能适用于多种伽玛逆转录病毒。这项研究的目的是将这一知识应用于减少基因递送系统中启动子/增强子插入对癌基因激活的影响。

 DESCRIPTION (provided by applicant): Murine leukemia virus preferentially integrates within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Our collaborative research has recently identified the host BET family of proteins, specifically the Brd 2, 3, and 4 proteins as interactors with the MLV IN and this interaction influences the integration into TSS and CpG islands. This provides the first window into our understanding of the mechanism of MLV target-site selection. This application studies the newly defined viral-host interaction between the MLV IN and BET family members. Four specific aims are built on two key observations. The first links the binding of Brd ET domains to the MLV IN C-terminal domain. The second indicates that IN proteins lacking the C-terminal 23 amino acids are no longer biased towards integration to TSS and CpG islands. Based on the solution structure of the MLV IN CTD from our laboratory, the first specific aims at defining the NMR structure of the MLV IN CTD:Brd3 ET complex. Additional structural analysis of the CTD in complex with the crossbone DNA integration intermediate will be pursued. The second specific aim applies mutational and biochemical assays to define the primary as well as potential secondary Brd binding sites. The controversial role of the IN CCD in binding BET proteins will be examined. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. The third specific aim utilizes next-generation sequencing and bioinformatics to probe a panel of IN proteins for their target-site preferences, both locally at the site of integraion as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. The final specific aim applies this information towards developing targeted integrating vectors. The research translates our strong knowledge of MLV IN towards rapidly understanding this interaction with the host target cell. The research addresses one of the outstanding questions relating to the mechanism of targeting MLV integration into promoter regions. These studies have the potential to be applicable to multiple gammaretroviruses. The goal of the research is to apply this knowledge towards decreasing the effects of promoter/enhancer insertions on oncogene activation in gene delivery systems.