Regulation And Function Of Retroelements

逆转录因子的调控和功能

• 批准号: 9550300
• 负责人: Henry L. LEVIN
• 金额: $ 84.23万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/9550300
• 关键词: Acquired Immunodeficiency Syndrome; Address; Aging; Animal Model; Antiviral Therapy; Behavior; Binding; Biological Assay; Biological Models; Cell Nucleus; Cells; Centromere; ChIP-seq; Chromatin Structure; Chromosomes; Collection; Complementary DNA; Complex; DNA; DNA Binding; DNA Polymerase II; DNA Polymerase III; DNA Repair; DNA Transposable Elements; DNA sequencing; DNA-Binding Proteins; Data; Defect; Disease; Distant; Drops; Elements; Enhancers; Environment; Eukaryota; Exhibits; Family; Fission Yeast; Frequencies; Gene Deletion; Gene Expression; Gene Transduction Agent; Genes; Genetic; Genetic Transcription; Genome; Genomics; HIV; HIV-1; Immunotherapy; Individual; Integrase; Integration Host Factors; Laboratories; Lead; Learning; Lentivirus Vector; Link; Long Terminal Repeats; Malignant Neoplasms; Mammals; Maps; Measures; Messenger RNA; Methods; Modeling; Molecular; Motivation; Mus; Mutation; Nucleotides; Oncogenic; Organism; Pathology; Pathway interactions; Pattern; Play; Positioning Attribute; Process; Promoter Regions; Property; Proteins; Proto-Oncogenes; Publishing; RNA; Regulation; Regulator Genes; Retroelements; Retrotransposon; Retroviral Vector; Retroviridae; Reverse Transcription; Risk; Role; Saccharomyces cerevisiae; Safety; Schizosaccharomyces; Sequence Analysis; Shapes; Side; Site; Specificity; Stress; Study models; System; T-Cell Proliferation; Terminator Codon; Testing; Therapy trial; Transcription Factor TFIIIB; Transfer RNA; Transport Vesicles; Vesicle Transport Pathway; Viral; Viral Vector; Virus; Yeasts; base; biological adaptation to stress; cancer therapy; cost; density; env Gene Products; gene therapy; genome-wide analysis; improved; integration site; interest; leukemia; novel strategies; nucleocytoplasmic transport; particle; pathogen; preference; promoter; repaired; response; telomere; tumor; type C retroviruses; vector

Diseases such as AIDS and leukemia caused by retroviruses have intensified the need to understand the mechanisms of retrovirus replication. One of our objectives is to understand how retroviral cDNAs are integrated into the genome of infected cells. Because of their similarities to retroviruses, long terminal repeat (LTR)-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. We are particularly interested in Tf1 because its integration exhibits a strong preference for pol II promoters. This choice of target sites is similar to the strong integration preferences human immunodeficiency virus 1 (HIV-1) and murine leukemina virus (MLV) have for pol II transcription units. Currently, it is not clear how these viruses recognize their target sites. We therefore study the integration of Tf1 as a model system with which we hope to uncover mechanisms general to the selection of integration sites. An understanding of the mechanisms responsible for targeted integration could lead to new approaches for antiviral therapies and to improvements in the application of viral vectors in gene therapy. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of S. pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors. Transposable elements constitute a substantial fraction of the eukaryotic genome and as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress response genes. The clustering of integration in specific promoters suggests Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment. We found that Sap1, an essential DNA binding protein, plays an important role in Tf1 integration. A mutation in Sap1 resulted in a 10-fold drop in Tf1 transposition and measures of transposon intermediates supports the argument that the defect occurred in the process of integration. Published ChIP-Seq data of Sap1 binding combined with high-density maps of Tf1 integration that measure independent insertions at single nucleotide positions show that 73.4% of all integration occurred at genomic sequences bound by Sap1. This represents high selectivity since Sap1 binds just 6.8% of the genome. A genome-wide analysis of promoter sequences revealed that Sap1 binding and amounts of integration correlate strongly. More importantly, an alignment of the DNA binding motif of Sap1 revealed integration clustered on both sides of the motif and showed high levels specifically at positions +19 and -9. These data indicate that Sap1 contributes to the efficiency and position of Tf1 integration. We continue our studies of Sap1 to determine its role in regulating the expression of the promoters it binds. Transposable elements (TEs) are common constituents of centromeres. However, it is not known what causes this relationship. Schizosaccharomyces japonicus contains 10 families of Long Terminal Repeat (LTR)-retrotransposons and these elements cluster in centromeres and telomeres. In the related yeast, Schizosaccharomyces pombe LTR-retrotransposons Tf1 and Tf2 are distributed in the promoter regions of RNA pol II transcribed genes. Sequence analysis of TEs indicates that Tj1 of S. japonicus is related to Tf1 and Tf2, and uses the same mechanism of self-primed reverse transcription. Thus, we wondered why these related retrotransposons localized in different regions of the genome. To characterize the integration behavior of Tj1 we expressed it in S. pombe. We found Tj1 was active and capable of generating de novo integration in the chromosomes of S. pombe. The expression of Tj1 is similar to Type C retroviruses in that a stop codon at the end of Gag must be present for efficient integration. 17 inserts were sequenced, 13 occurred within 12 bp upstream of tRNA genes and 3 occurred at other RNA pol III transcribed genes. The link between Tj1 integration and RNA pol III transcription is reminiscent of Ty3, an LTR-retrotransposon of Saccharomyces cerevisiae that interacts with TFIIIB and integrates upstream of tRNA genes. The integration of Tj1 upstream of tRNA genes and the centromeric clustering of tRNA genes in S. japonicus demonstrate that the clustering of this TE in centromere sequences is due to a unique pattern of integration. Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the specificity of integration site selection. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a unique genetic assay that measures cDNA present in the nucleus, we could identify factors that contribute to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they suggest the pathways that repair the single stranded gaps opposite integration sites are the same in diverse eukaryotes.

逆转录病毒引起的艾滋病和白血病等疾病加剧了了解逆转录病毒复制机制的需求。我们的目标之一是了解如何将逆转录病毒cDNA整合到感染细胞的基因组中。由于它们与逆转录病毒的相似性，长期重复（LTR） - 返回转座子是逆转录病毒复制的重要模型。在我们的实验室研究的逆转录座子是裂变酵母菌酵母酸酯POMBE的TF1元素。我们对TF1特别感兴趣，因为它的整合表现出对Pol II启动子的强烈偏爱。 目标位点的这种选择类似于POL II转录单元的强大整合偏好人类免疫缺陷病毒1（HIV-1）和鼠白血病病毒（MLV）的HAM。目前，尚不清楚这些病毒如何识别其目标部位。因此，我们研究了TF1作为模型系统的整合，我们希望将其揭示为一般选择集成位点的机制。对负责靶向整合的机制的理解可能会导致抗病毒疗法的新方法，并改善病毒载体在基因疗法中的应用。 DNA测序的非凡能力可以创建超密集的整合图，用于研究位置整合的机制。不幸的是，检测到的插入站点数量的巨大增加是由于不知道哪些位置是罕见目标以及哪些位置持续插入的成本。为了解决这个问题，我们开发了序列号系统，这是一种TE标记方法，可测量单核苷酸位置的整合频率。 我们测序了100万个逆转座子TF1在S. pombe的基因组中的插入，并获得了每个单个位置的频率的首个集成谱。 单个核苷酸的整合水平在两个数量级上有所不同，并揭示了序列识别在定位整合中起关键作用。序列号系统是一种通用方法，可用于确定逆转录病毒和基因治疗载体的精确整合图。 转座元素占真核基因组的很大一部分，因此，与他们的宿主建立了复杂的关系，既是对抗性又依赖的。为了最大程度地减少对细胞基因的损害，其具有将整合到低重要性序列的机制。 然而，精神分裂症pombe的逆转座子TF1与应激反应基因的启动子序列的令人惊讶的偏差相结合。 特定启动子中整合的聚类表明，TF1具有一种靶向机制，对于对环境变化的进化适应至关重要。我们发现SAP1是必不可少的DNA结合蛋白，在TF1整合中起重要作用。 SAP1中的一个突变导致TF1换位的10倍下降，而转座中间体的度量支持以下论点：缺陷发生在整合过程中。 SAP1结合的已发布的芯片seq数据与TF1整合的高密度图相结合，测量在单核苷酸位置的独立插入表明，所有整合的73.4％发生在由SAP1结合的基因组序列上。 这代表了高选择性，因为SAP1仅结合基因组的6.8％。 对启动子序列的全基因组分析表明，SAP1结合和整合量密切相关。更重要的是，SAP1的DNA结合基序的比对揭示了在基序的两侧聚集的整合，并在位置+19和-9的位置特别显示高水平。这些数据表明SAP1有助于TF1整合的效率和位置。我们继续对SAP1进行研究，以确定其在调节其结合的启动子表达中的作用。 转座元素（TES）是丝粒的常见成分。但是，尚不知道是什么原因导致这种关系。 精神分裂症Japonicus包含10个长末期重复（LTR） - 返回转座子的家族，这些元素聚集在centromeres and端粒中。在相关的酵母中，schizosaccharomyces pombe ltr-Retransposons tf1和tf2分布在RNA Pol II转录基因的启动子区域中。 TES的序列分析表明，Japonicus链球菌的TJ1与TF1和TF2有关，并使用了自prifed逆转录的相同机理。因此，我们想知道为什么这些相关的逆转座子位于基因组的不同区域。为了表征TJ1的整合行为，我们在S. pombe中表达了它。 我们发现TJ1具有活性，能够在S. pombe的染色体中产生从头积分。 TJ1的表达与C型逆转录病毒相似，因为必须存在GAG末端的终止密码子以进行有效整合。测序了17个插入物，在tRNA基因上游的12 bp中发生了13件，在其他RNA POL III转录基因上发生了3个。 TJ1整合与RNA POL III转录之间的联系让人联想到Ty3，Ty3是一种与TFIIIB相互作用并整合tRNA基因上游的糖含量的Ltr-Retrotrantposon。 tRNA基因上游的TJ1整合和japonicus链球菌中tRNA基因的丝粒聚类表明，该TE在中心仪序列中的聚类是由于独特的整合模式所致。 逆转录病毒和长时间重复（LTR） - 返回转座子具有不同的积分位点模式。 基因治疗中使用的基于逆转录病毒的载体的致癌潜力取决于整合位点的选择。将精神分裂症的LTR-返回旋转型TF1作为模型进行了研究，因为它将其集成到应激反应基因的启动子中。尽管由逆转录病毒和LTR-返回转座子编码的积分（INS）负责催化cDNA插入宿主基因组中，但人们认为，积​​分位点选择的特异性是必需的。我们通过促进TF1整合的宿主因子的全基因组筛选来检验这一假设。通过将换位的测定与测量核中存在的cDNA的独特遗传测定法相结合，我们可以确定有助于整合的因素。 我们利用该测定法测试了带有单基因缺失的3,004 S. POMBE菌株的集合。使用这些筛选和TF1蛋白的免疫印迹测量，我们确定了促进整合的61个基因。整合因子参与了一系列过程，包括核运输，转录，mRNA加工，囊泡运输，染色质结构和DNA修复。出乎意料的是，我们先前发现了许多我们确定的途径，以促进酿酒酵母中LTR-返回转座子Ty1和Ty3的整合，表明宿主因子对整合的贡献在较远相关的生物体中很常见。 DNA修复因子特别令人感兴趣，因为它们表明在不同的真核生物中修复与整合位点相反的单个链间隙的途径相同。