Regulation And Function Of Retroelements

逆转录因子的调控和功能

基本信息

批准号：
8553862
负责人：
Henry L. LEVIN
金额：
$ 151.33万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/8553862
关键词：
Acquired Immunodeficiency Syndrome Address Adopted Alleles Animal Model Antiviral Therapy Binding Biological Biological Assay Biological Models Biology Cell Nucleus Cell Survival Cell division Cells Collaborations Complementary DNA Coupled DNA DNA Polymerase II DNA Polymerase III DNA Sequence DNA Transposons Data Diploidy Disease Elements Enhancers Essential Genes Eukaryota Event Evolution Exhibits Family Fission Yeast Gene Expression Generations Genes Genetic Genetic Transcription Genome Genomics Goals Growth Gypsies HIV-1 Haploidy Heating Heterochromatin Histones Hot Spot Human Immunologic Deficiency Syndromes Indium Individual Infant Integrase Integration Host Factors Laboratories Lead Ligation Long Terminal Repeats Malignant Neoplasms Maps Measures Mediating Methods Modeling Molecular Motivation Murine leukemia virus Mus Musca domestica Ontology Open Reading Frames Oxidative Stress Parasites Pathology Patients Pattern Phenotype Plasmids Play Property Regulation Reporting Research Response Elements Retroelements Retrotransposon Retroviral Vector Retroviridae Reverse Transcription Risk Role Saccharomyces cerevisiae Safety Site Specificity Stress Structure Study models Technology Testing Transcript Transcription Coactivator Transcription Initiation VP 16 Virus Work Yeasts base biological adaptation to stress density env Gene Products gene discovery gene function gene induction gene therapy genome-wide improved interest leukemia member mutant novel novel strategies particle preference promoter research study restriction enzyme transcription factor vector

项目摘要

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. A specific goal of our research is to identify the mechanism that directs integration to regions upstream of ORFs. To study insertion patterns in specific genes, a target plasmid assay was developed. Integration into the promoter of fbp1 clustered within 10 bp of a transcription enhancer called upstream activating sequence 1 (UAS1). Integration into the promoter of fbp1 depended on UAS1 sequence and Atf1p, a transcription activator that binds UAS1. To identify the key determinants responsible for targeting integration in the fbp1 promoter we conducted an extensive study of the promoter sequences. Here we report that two discrete target windows close to UAS1 were the only sequences in the promoter required for the pattern of integration. These two target windows functioned independently of each other and each one was found to be sufficient to function as an efficient target of integration. Although Atf1p is necessary for directing integration to UAS1, it may be that by activating transcription, Atf1p induces subsequent steps of transcription that are more directly responsible for directing integration. If the role of Atf1p in integration were indirect, other factors that promote fbp1 transcription would also influence integration at this promoter. However, other known transcription factors that mediate fbp1 transcription, Pcr1p, Rst2p and Tup11p/Tup12p were found not to contribute to integration at UAS1. UAS2 is an independent enhancer in the promoter of fbp1 and was not a target of integration. Nevertheless, we found UAS2 did promote efficient transcription of fbp1. In addition, we found a synthetic promoter induced by lexA fused to an activator, VP16, was not a target of Tf1 integration. These data indicate that transcription activity of a promoter is not sufficient to mediate integration. Instead, the data indicate Atf1p plays a direct and specific role in targeting integration to UAS1 of the fbp1 promoter. The result that integration of Tf1 is directed to the promoters of genes raises several key questions about the biology of Tf1 integration. Are all promoters recognized equally or is integration directed to specific sets of promoters. If specific sets of promoters are preferred targets, what distinguishes the preferred promoters from those not recognized by Tf1. To address these questions we sequenced large numbers of insertions throughout the genome of S. pombe using the new methods for ultra high throughput sequencing. We performed four independent transposition experiments using haploids and diploids and two different restriction enzymes (Mse I or Hpy CH4 IV) to digest the genomic DNA. All together BLAST results identified 73,125 independent Tf1 integration events. The vast majority of insertions clustered within 500 bp upstream of ORFs confirming the preference for promoter sequences observed in the target plasmid assays and the small numbers of genomic insertions previously characterized. Analysis of integration levels in promoters revealed that Tf1 integration is high in approximately 1,000 promoters. A comparison of independent experiments revealed that we obtained a reproducible and saturated measure of the integration in each of the 5,000 promoters of S. pombe. The results of the gene ontology analysis revealed that genes regulated by environmental stress were favored targets of integration. The strongest correlations were seen with promoters induced by osmotic and oxidative stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests the intriguing possibility that Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress. To determine the biological significance of Tf1 integration we took advantage of saturated maps of insertion activity and studied how integration at hot spots effected the expression of the adjacent genes. Our study of 32 genes at common sites of insertion revealed that Tf1 integration did not reduce gene expression. Importantly, the insertions activated the expression of 6 transcripts corresponding to 19% of the genes tested. Our analysis of transcripts revealed Tf1 induced genes by inserting enhancer activity. Interestingly, the enhancer activity of Tf1 could be limited by Abp1, a host surveillance factor that sequesters transposon sequences into structures containing histone deacetylases. We found the Tf1 promoter was activated by heat treatment and remarkably, only genes that themselves were induced by heat could be activated by Tf1 integration, suggesting a synergy of Tf1 enhancer sequence with the stress response elements of target promoters. We propose that the integration preference of Tf1 for the promoters of stress response genes and the ability of Tf1 to induce these genes co-evolved to enhance the survival of cells under stress. With the introduction of new deep sequencing technology it is now possible to sequence many millions of transposon insertions in a single experiment. We tested whether Illumina sequencing could be used to generate a dense profile of transposon insertions that would reveal which genes are required for cell division. For this experiment we used a haploid strain of S. pombe and Hermes, a DNA transposon from the housefly. In previous work we found that the Hermes transposon was highly active in S. pombe and that a large fraction of the insertions occurred in ORFs. We predicted that in actively growing cultures, Hermes insertions would not be tolerated in essential ORFs. We induced Hermes transposition in a large culture S. pombe that was grown for 80 generations. With ligation mediated PCR and Illumina sequencing we were able to sequence 360,513 independent insertion events. On average, this represented one insertion for every 29 bp of the S. pombe genome. An analysis of integration density revealed that the ORFs largely separated into two classes, one with high numbers of insertions and another with much lower numbers. In collaboration with a group that deleted each of the genes of S. pombe, we found the ORFs with low numbers of Hermes insertion corresponded to the essential genes. The ORFs with higher integration densities were in genes classified as nonessential. These results validated integration profiling as a new method for identifying genes with essential function. Importantly, by applying specific conditions of selection during growth, this method can be adopted to identify genes that contribute to a wide variety of functions.

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。对负责有针对性整合的机制的理解可能会导致抗病毒疗法的新方法。我们研究的一个具体目标是确定将整合到ORF上游地区的机制。为了研究特定基因中的插入模式，开发了靶质粒测定。集成到FBP1的启动子中，这些启动子聚集在10 bp的转录增强子中，称为上游激活序列1（UAS1）。集成到FBP1的启动子中取决于UAS1序列和ATF1P，ATF1P是结合UAS1的转录激活剂。为了确定负责靶向FBP1启动子中整合的关键决定因素，我们对启动子序列进行了广泛的研究。在这里，我们报告说，靠近UAS1的两个离散目标窗口是集成模式所需的启动子中唯一的序列。这两个目标窗口彼此独立起作用，每个目标窗口都足以充当积分的有效目标。尽管ATF1P对于将整合引入UAS1是必要的，但可能是通过激活转录，ATF1P引起了随后的转录步骤，而转录步骤更直接地指导集成。如果ATF1P在整合中的作用是间接的，那么促进FBP1转录的其他因素也会影响该启动子的整合。然而，发现其他已知的转录因子介导FBP1转录，PCR1P，RST2P和TUP11P/TUP12P不促进UAS1的整合。 UAS2是FBP1启动子的独立增强子，不是集成的目标。然而，我们发现UAS2确实促进了FBP1的有效转录。此外，我们发现由Lexa诱导的与激活剂VP16诱导的合成启动子不是TF1整合的目标。这些数据表明启动子的转录活性不足以介导整合。取而代之的是，数据表明ATF1P在靶向与FBP1启动子的UAS1的靶向中起着直接而特定的作用。 TF1的整合针对基因的启动子提出了有关TF1整合生物学的几个关键问题。都是公认的启动子，或者是针对特定启动子组的集成。如果特定的启动子是首选目标，则区分首选启动子与未经TF1认可的启动子的区别。为了解决这些问题，我们使用新的超高吞吐量测序的新方法对整个S. pombe的整个基因组进行了大量插入。我们使用单倍体和二倍体以及两种不同的限制酶（MSE I或HPY CH4 IV）进行了四个独立的换位实验，以消化基因组DNA。爆炸结果总共确定了73,125个独立的TF1整合事件。绝大多数插入聚集在ORF上游的500 bp之内，证实了在靶质粒测定中观察到的启动子序列的偏好以及先前表征的少量基因组插入。启动子中整合水平的分析表明，大约1,000个启动子中TF1的整合很高。对独立实验的比较表明，我们在5,000个pombe的5,000个启动子中的每个启动子中获得了可再现和饱和度量的量度。基因本体分析的结果表明，受环境压力调节的基因是融合的偏爱靶标。与渗透和氧化应激诱导的启动子观察到最强的相关性。这种靶向应力反应基因的靶向以及TF1调节相邻基因表达的能力的靶向表明，当细胞暴露于环境应激时，TF1可能会提高TF1的生存率。为了确定TF1整合的生物学意义，我们利用了插入活性的饱和图，研究了热点的整合如何影响相邻基因的表达。我们对插入常见位点的32个基因的研究表明，TF1整合并未降低基因表达。重要的是，插入激活了6个对应于19％基因的转录本的表达。我们对转录本的分析揭示了TF1通过插入增强剂活性来诱导的基因。有趣的是，TF1的增强剂活性可能受到ABP1的限制，ABP1是一种宿主监测因子，将转座子序列隔离到含有组蛋白脱乙酰基酶的结构中。我们发现TF1启动子是通过热处理激活的，而且只有TF1整合才能激活其本身是由热量诱导的基因，这表明TF1增强子序列的协同作用与靶启动子的应力响应元素的协同作用。我们提出，TF1对应激反应基因的启动子的整合偏好以及TF1诱导这些基因共同发展以增强细胞在应激下的生存的能力。通过引入新的深层测序技术，现在可以在一个实验中对数百万个转座子插入进行测序。我们测试了Illumina测序是否可以用于生成转座插入的密集曲线，这些插入将揭示细胞分裂所需的基因。在此实验中，我们使用了单倍体菌菌和爱马仕（Hermes）的单倍体菌株，Hermes是来自Housefly的DNA转座子。在先前的工作中，我们发现爱马仕（Hermes）转座子在庞贝（S. pombe）中高度活跃，很大一部分插入发生在ORF中。我们预测，在积极成长的文化中，爱马仕的插入将在基本的ORF中宽容。我们诱导了爱马仕（Hermes）在大型培养的S. pombe中的旋转，该培养物生长了80代。通过连接介导的PCR和Illumina测序，我们能够对360,513个独立的插入事件进行测序。平均而言，这代表了S. pombe基因组的每29 bp的一个插入。对整合密度的分析表明，ORF在很大程度上分为两个类别，一个插入数量较高，数量较低。与一个删除了S. pombe基因的小组合作，我们发现HERMES插入数量少的ORF与基本基因相对应。具有较高积分密度的ORF在分类为非必需的基因中。这些结果验证了集成概况作为识别具有基本功能的基因的新方法。重要的是，通过在增长过程中应用特定的选择条件，可以采用此方法来识别有助于各种功能的基因。