Regulation And Function Of Retroelements

逆转录因子的调控和功能

基本信息

批准号：
10686720
负责人：
Henry L. LEVIN
金额：
$ 136.19万
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/10686720
关键词：
Address Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animal Model Binding Binding Sites Biological Assay Brain Cell Nucleus Cell physiology Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Complex DNA DNA Insertion Elements DNA Transposable Elements Data Disease Dissociation Elements Event Family Fission Yeast Frequencies Future Gene Expression Genes Genetic Diseases Genetic Recombination Genetic Transcription Genetic Variation Genome Genomic DNA Genomics Genotype Genotype-Tissue Expression Project Goals HIV-1 HIV-1 integrase Health Heat-Shock Response Human Human Genetics Human Genome Hypoxia Individual Infection Integrase Integrase Inhibitors Lead Long Interspersed Elements Long Terminal Repeats Luciferases Maps Mental disorders Migraine Modeling Molecular Mutation Nature Northern Europe Parkinson Disease Pathway interactions Patients Pattern Play Positioning Attribute Predisposition Proxy Publishing Purines Quantitative Trait Loci RAD52 gene Regulation Regulatory Element Reporter Reporting Research Residual state Resolution Retroelements Retrotransposition Retrotransposon Retroviridae Reverse Transcription Risk Role Sampling Schizophrenia Shapes Short Interspersed Nucleotide Elements Single Nucleotide Polymorphism Site Source Stress Study models Testing Tissues Trans-Activators United States National Institutes of Health Utah Variant Viral Virus Western Europe Work Yeasts brain tissue causal variant data modeling env Gene Products epigenomics gene network gene regulatory network genetic association genetic variant genome editing genome wide association study genome-wide homologous recombination human disease human population study induced pluripotent stem cell insertion/deletion mutation integration site markov model nerve stem cell nervous system disorder particle pathogen preference promoter response trait transcriptome transcriptome sequencing tumor viral DNA

Address Aging Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animal Model Binding Binding Sites Biological Assay Brain Cell Nucleus Cell physiology Cells Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Complex DNA DNA Insertion Elements DNA Transposable Elements Data Disease Dissociation Elements Event Family Fission Yeast Frequencies Future Gene Expression Genes Genetic Diseases Genetic Recombination Genetic Transcription Genetic Variation Genome Genomic DNA Genomics Genotype Genotype-Tissue Expression Project Goals HIV-1 HIV-1 integrase Health Heat-Shock Response Human Human Genetics Human Genome Hypoxia Individual Infection Integrase Integrase Inhibitors Lead Long Interspersed Elements Long Terminal Repeats Luciferases Maps Mental disorders Migraine Modeling Molecular Mutation Nature Northern Europe Parkinson Disease Pathway interactions Patients Pattern Play Positioning Attribute Predisposition Proxy Publishing Purines Quantitative Trait Loci RAD52 gene Regulation Regulatory Element Reporter Reporting Research Residual state Resolution Retroelements Retrotransposition Retrotransposon Retroviridae Reverse Transcription Risk Role Sampling Schizophrenia Shapes Short Interspersed Nucleotide Elements Single Nucleotide Polymorphism Site Source Stress Study models Testing Tissues Trans-Activators United States National Institutes of Health Utah Variant Viral Virus Western Europe Work Yeasts brain tissue causal variant data modeling env Gene Products epigenomics gene network gene regulatory network genetic association genetic variant genome editing genome wide association study genome-wide homologous recombination human disease human population study induced pluripotent stem cell insertion/deletion mutation integration site markov model nerve stem cell nervous system disorder particle pathogen preference promoter response trait transcriptome transcriptome sequencing tumor viral DNA

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项目摘要

Identification of an integrase-independent pathway of retrotransposition Despite the central role of integration in the propagation of retroviruses, important questions remain about residual insertion that occurs in the absence of IN activity. Mutations in the catalytic residues of HIV-1 integrase (IN) produce residual infectious titer, typically with a 3 to 4-log decrease. However, in continuous cultures of HIV-1 lacking IN activity, insertion efficiency can be as high as 0.2-0.8% of wild type virus. These results indicate that retroviruses possess a secondary, IN-independent pathway, that incorporates viral DNA into the host genome. Since IN-independent infections could compromise the treatment of HIV-1 patients with IN inhibitors, it is important to identify the nature of this pathway. Long terminal repeat (LTR) retrotransposons are important models of retroviruses due to structural and mechanistic similarities. We found that the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe retains 5% insertion activity in the absence of IN. Genome-wide insertion profiles of Tf1 lacking IN (Tf1-INfs) were significantly different from that of Tf1 expressing active IN. DNA logo analysis showed that the sequences downstream of the Tf1-INfs insertion sites had a prominent bias of ATAAC and upstream flanks showed a preference of CAA. Interestingly, the downstream logo matches that of the primer binding site (PBS), an 11 bp sequence retained after reverse transcription on the 3end of the +strand cDNA. The CAA matches the last three bp of the poly purine tract (PPT), that is retained on the 3 end of the -strand cDNA. The PBS and PPT preferences suggested that these single-stranded sequences contributed to insertion through homologous recombination (HR). Transposition assays revealed the insertions occurred through Rad52-dependent single-strand annealing (SSA), as Rad51 was dispensable. The rad52-R45A mutation, which specifically abolishes SSA activity of Rad52, significantly reduced the frequency of Tf1-INfs insertions and resulted in dissociation of Rad52 from Tf1 cDNA. These data indicate that Rad52 plays a critical role in IN independent insertions by binding to the ends of the cDNA causing recombination with sequences similar to PBS and PPT. S. pombe contains 13 pre-existing copies of Tf2, an LTR-retrotransposon with PBS and PPT sequence identical to Tf1. We tested whether Tf2 was a significant site of IN independent insertion by re-analyzing raw sequence reads from Tf1-INfs insertions that otherwise would be discarded because they map to multiple positions. Indeed, we found that 70% of the insertion activity mapped to the PBS of Tf2. The insertion sequence of individual strains expressing Tf1-INfs revealed insertions occurred at existing Tf2s and resulted in tandem elements. Importantly, tandem copies of Tf2 regularly form in wild isolates of S. pombe. Our efforts to determine whether IN independent events occur naturally showed cultures with continuing expression of WT Tf1 produced insertions that were predominantly IN independent. These data demonstrate that Tf1 possesses two efficient insertion pathways, one relies on IN and the other is IN independent but requires Rad52. Significantly, we found in previously published data of HIV-1 IN independent insertions that five of 69 sites had strong similarity to the HIV-1 PBS. Together, these results indicate that homology dependent SSA provides a significant pathway of IN independent insertion. Retrotransposon insertions associated with risk of neurologic and psychiatric diseases Genetic variation can directly cause or increase susceptibility to neurologic and psychiatric diseases. Genome-wide association studies (GWAS) have been very useful in identifying single nucleotide polymorphisms (SNPs) associated with common and complex human diseases. However, identifying causal genetic variants remains challenging because most disorders are influenced by many loci and SNPs do not typically provide adequate resolution to pinpoint causal genes. In addition, GWAS often do not directly identify structural variants such as TE insertions which can have substantial impact on gene expression. TE activity contributes to disease with reports of more than 124 cases of TE insertions that result in human genetic diseases, and elevated transposition in brain is observed in neurologic diseases. GWAS SNPs can serve as proxies for genomic insertions and deletions because these structural variants are more likely to alter gene expression than SNPs. Importantly, polymorphic TEs map next to disease-associated SNPs more frequently than expected by chance. Therefore, we hypothesized that polymorphic TEs highly associated with GWAS SNPs of neurologic and psychiatric diseases are potential causative variants of those diseases. We took advantage of the 17,000 polymorphic TEs mapped by the 1,000 Genomes Project to determine what role TEs may have in neurologic and psychiatric diseases. For our analysis we reviewed GWAS of neurologic and psychiatric diseases including amyotrophic lateral sclerosis, migraine, schizophrenia, Parkinson's disease, Alzheimer's disease, and identified 753 Trait Associated SNPs (TASs) with P < 106. We calculated and ranked the levels of genetic association between TASs and cataloged polymorphic TEs. This generated 76 candidate TEs that are highly associated with a TAS. From these variants, we identified causal candidate TEs by searching for 1) TEs inserted in regulatory chromatin active in brain tissues as determined by Hidden Markov modeling of data from the NIH Roadmap Epigenomics Consortium, and 2) TEs associated with altered gene expression in brain tissues using RNA-seq data from the Genotype-Tissue Expression (GTEx) project to identify expression Quantitative Trait Loci (eQTLs). We further validated association level of TAS and candidate TEs by genotyping polymorphic TE insertions using genomic DNA from a 30-trio reference panel of CEPH Utah residents with ancestry from northern and western Europe (CEU) HapMap samples from the 1000 Genome Project. In summary, we found 10 polymorphic TE insertions that are important causal candidates of neurologic and psychiatric disorders. Among them, lead TE insertions were tested for regulatory effects in human neural stem cells by luciferase reporter assay, and 5 of 6 tested caused significant changes in the activity of a minimal promoter. Future studies include testing the direct impact of the causal candidate TEs by CRISPR/Cas genome editing of human iPSCs.

鉴定逆转录词的独立酶独立途径尽管整合在逆转录病毒的传播中的核心作用，但在没有活性的情况下，仍然存在有关残留插入的重要问题。 HIV-1积分酶（IN）催化残基的突变产生残留的传染性滴度，通常降低3至4型。然而，在缺乏活性的HIV-1的连续培养物中，插入效率可能高达野生型病毒的0.2-0.8％。这些结果表明，逆转录病毒具有将病毒DNA纳入宿主基因组的次级，独立的途径。由于内部依赖性感染可能会损害抑制剂中HIV-1患者的治疗，因此鉴定该途径的性质很重要。由于结构和机械的相似性，长期重复（LTR）逆转录子是逆转录病毒的重要模型。我们发现，在没有In的情况下，schizosacchomyces pombe的LTR-返回旋转TF1保留了5％的插入活性。缺乏（TF1-INF）的TF1的全基因组插入曲线与表达活性的TF1显着不同。 DNA徽标分析表明，TF1-INFS插入位点下游的序列具有ATAAC的显着偏见，并且上游侧面显示出CAA的偏好。有趣的是，下游徽标与引物结合位点（PBS）的徽标匹配，这是在 +链cDNA的3端上逆转录后保留的11 bp序列。 CAA与保留在-strand cDNA的3端的聚嘌呤道（PPT）的最后三个BP匹配。 PBS和PPT偏好表明，这些单链序列通过同源重组（HR）插入。换位测定表明，由于RAD51是可分配的，这是通过RAD52依赖性单链退火（SSA）发生的。 RAD52-R45A突变特别废除了RAD52的SSA活性，显着降低了TF1-INFS插入的频率，并导致Rad52与TF1 cDNA的解离。这些数据表明，Rad52通过与cDNA的末端结合，与类似于PBS和PPT的序列结合，在独立插入中起着至关重要的作用。 S. pombe包含13份TF2的预先存在的副本，TF2是一种具有PBS和PPT序列与TF1相同的LTR返回跨跨型。我们通过重新分析从TF1-INFS插入的原始序列读取的原始序列来测试TF2是否是独立插入的重要位点，否则将被丢弃，因为它们映射到多个位置。确实，我们发现映射到TF2 PBS的70％的插入活性。表达TF1-INF的个体菌株的插入顺序显示在现有的TF2S处发生插入，并导致串联元素。重要的是，TF2的串联副本在pombe的野生分离株中定期形成。我们确定在独立事件中是否发生的努力自然表明培养物，WT TF1的持续表达产生的插入主要是独立的。这些数据表明，TF1具有两个有效的插入途径，一个依赖于IN，另一个是独立的，但需要RAD52。值得注意的是，我们在独立插入的HIV-1的先前发表的数据中发现，69个站点中有5个与HIV-1 PBS具有很强的相似性。总之，这些结果表明同源性依赖性SSA提供了独立插入的重要途径。与神经和精神病风险相关的逆转录座插入遗传变异可以直接引起或增加对神经系统疾病和精神疾病的敏感性。全基因组关联研究（GWAS）在鉴定与常见和复杂人类疾病相关的单核苷酸多态性（SNP）方面非常有用。但是，识别因果遗传变异仍然具有挑战性，因为大多数疾病受许多基因座的影响，而SNP通常不能提供足够的分辨率来查明因果基因。此外，GWA通常不会直接识别可能对基因表达产生重大影响的TE插入等结构变异。 TE活性导致了疾病，其中有124例TE插入导致人类遗传疾病的报道，并且在神经系统疾病中观察到大脑中的转置升高。 GWAS SNP可以用作基因组插入和缺失的代理，因为这些结构变异比SNP更可能改变基因表达。重要的是，与偶然相关的疾病相关SNP旁边的多态性TES图。因此，我们假设多态TE与神经系统疾病和精神病的GWAS SNP高度相关是这些疾病的潜在病因变异。我们利用了1,000个基因组项目映射的17,000个多态性TE，以确定TE在神经系统疾病和精神疾病中的作用。在我们的分析中，我们回顾了神经系统和精神病疾病的GWA，包括肌萎缩性侧面硬化症，偏头痛，精神分裂症，帕金森氏病，阿尔茨海默氏病，并确定了753个特征相关的SNP（Tass），p <106。这产生了与TA高度相关的76个候选TE。 From these variants, we identified causal candidate TEs by searching for 1) TEs inserted in regulatory chromatin active in brain tissues as determined by Hidden Markov modeling of data from the NIH Roadmap Epigenomics Consortium, and 2) TEs associated with altered gene expression in brain tissues using RNA-seq data from the Genotype-Tissue Expression (GTEx) project to identify expression Quantitative Trait Loci （eqtls）。我们通过使用来自北欧和西欧（CEU）HAPMAP样本的30个三重奏参考小组的基因组DNA来进一步验证TA和候选TES的关联水平。总而言之，我们发现10个多态性插入是神经系统和精神疾病的重要因果候选者。其中，通过荧光素酶报告基因测定法测试了铅插入在人类神经细胞中的调节作用，而6个测试中的5个导致最小启动子的活性发生显着变化。未来的研究包括通过人IPSC的CRISPR/CAS基因组编辑来测试因果候选TE的直接影响。