Interactions of MuLV IN with host proteins and DNA

MuLV IN 与宿主蛋白和 DNA 的相互作用

基本信息

批准号：
9267487
负责人：
MONICA J ROTH
金额：
$ 37.68万
依托单位：
RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9267487
关键词：
Address Affect Amino Acids Binding Binding Sites Biochemical Bioinformatics Biological Assay C-terminal Catalytic Domain Cells Complex CpG Islands DNA DNA Integration DNA Structure DNA sequencing Disease Enhancers Epigenetic Process Family member Gammaretrovirus Gene Delivery Goals Histone Deacetylase Inhibitor Human Genome Integrase Integration Host Factors Knowledge Laboratories Ligand Binding Domain Link Modeling Murine leukemia virus Mutagenesis Mutation Nature Nucleic Acids Nucleosomes Oncogene Activation Oncogenic Pathogenesis Peptides Play Promoter Regions Protein Family Proteins Proviruses Research Role Site Structure Tail Tertiary Protein Structure Testing Transcription Initiation Site Translating Variant Viral Viral Proteins Viral Vector Virus Virus Integration Virus Replication base combinatorial driving force experimental study gene delivery system gene product inhibitor/antagonist insight integration site next generation next generation sequencing preference promoter public health relevance research study vector

项目摘要

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.

描述（由申请人提供）：鼠白血病病毒preped witin活跃的启动子区域RT站点（TSS）和CPG岛。整合地点与病毒的致癌潜力有关交付媒介与MLV相互作用，这种相互作用会影响TSS和CPG岛的整合。在两个关键观察中建立了四个特定的目标。基于我们实验室中CTD中MLV的溶液结构，将MLV的NMR结构定义为CTD：SBONE DNA Integration。将研究CCD在结合BET蛋白中的主要作用。将研究与已知ET域相互作用的竞争。在人类基因组中进行了靶位Y，对核小体结构中的目标DNA的识别应用于最终的特定目的。目标细胞解决了与启动子区域的靶向靶标有关的杰出问题。