Development and application of transposable element technology

转座元件技术的开发及应用

• 批准号: 10007509
• 负责人: Henry L. LEVIN
• 金额: $ 61.68万
• 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/10007509
• 关键词: Acids; Address; Alleles; Amino Acids; Animal Model; Bacteria; Binding Sites; Biology; C-terminal; Cells; Centromere; ChIP-seq; Chromatin; Chromosome Segregation; Clinical Trials; Complex; Coupled; DNA; DNA Transposable Elements; DNA sequencing; Data; Defect; Development; Disabled Persons; Epigenetic Process; Epitopes; Essential Genes; Eukaryota; Event; Excision; Exhibits; Fission Yeast; Gene Silencing; Genes; Genome; Goals; Growth; Heterochromatin; High-Throughput Nucleotide Sequencing; Histone H3; Histones; Homologous Protein; Individual; Libraries; Link; Lysine; Mating Types; Meiosis; Messenger RNA; Methods; Methyltransferase; Modeling; Molecular; Mutagenesis; Mutation; Nitrogen; Nuclear; Overlapping Genes; Pathogenicity; Pathway interactions; Phenotype; Phosphodiesterase I; Play; Polyadenylation; Polymerase; Positioning Attribute; Proteins; RNA; RNA Interference; RNA Processing; RNA-Binding Proteins; Reporter; Role; Saccharomyces cerevisiae; Small Interfering RNA; Structure; Technology; Testing; Transcript; Uracil; Yeasts; base; chromatin protein; deep sequencing; exosome; experimental study; gene discovery; gene function; gene therapy; genome integrity; genome-wide; histone methylation; mRNA Cleavage and Polyadenylation Factors; mutant; novel; novel strategies; recruit; tool; transcription termination; transcriptome sequencing; transposon sequencing

In eukaryotes the assembly of DNA into highly condensed heterochromatin is critical for a broad range of functions related to genome integrity. The methylation of histone H3 on lysine 9 (H3K9me) is central to the formation of heterochromatin by creating binding sites for a range of chromatin proteins important for silencing transposable elements, chromosome segregation, and epigenetic inheritance. S. pombe is an excellent model that has been used extensively to study the molecular mechanisms that generate and regulate heterochromatin. Centromeres, subtelomeres, and the mating-type region are packaged into constitutive heterochromatin while meiosis genes are silenced by facultative heterochromatin until cells are starved for nitrogen. Importantly, Clr4, the histone H3K9 methyltransferase is recruited to heterochromatin regions by multiple mechanisms. Constitutive heterochromatin results from RNAi factors that include Ago1 containing RNA-induced transcriptional silencing complex (RITS). Facultative heterochromatin at meiosis genes is independent of RNAi and relies on RNA elimination factors Red1, Mmi1, and the nuclear exosome. However, gaps exist in understanding how different processing activities function to target heterochromatin. A new approach for identifying gene function is the high throughput sequencing of integration profiles also known as Tn-Seq which identifies genes important for growth in selective conditions. Genes necessary to sustain growth under a specific condition do not tolerate insertions in that condition. Tn-seq has been applied to identify pathogenic genes in bacteria. However, we developed this method for identifying essential genes in yeast and subsequently others have applied the strategy. With the goal of identifying novel factors important for heterochromatin we produced dense profiles of integrations using the Hermes transposable element and a silencing reporter (ura4) positioned in the outer repeats of centromere 1. Inserts that disrupted genes important for heterochromatin activated ura4 and thus the cells were unable to grow when passaged in 5-fluoroorotic acid (FOA). Genes with established roles in heterochromatin assembly had significantly fewer insertions in the cells with the centromere reporter otr1R::ura4 relative to cells lacking the reporter. The list of candidates contained a total of 199 genes and importantly, 65 are known to be essential for viability. These essential genes were candidates because they tolerated many insertions in their 3sequences that reduced heterochromatin but not viability. The high number of essential genes is significant in that most proteins found to be important for heterochromatin are identified in screens of deletion strains that cannot include essential genes. The 199 candidates showed highly significant enrichments for functions in silencing at centromere outer repeats and included all four factors that produce siRNA. Other RNA processing factors were identified that were not previously linked to heterochromatin structure. Strikingly, four of the RNA processing candidates form an interaction module of canonical mRNA polyadenylation and cleavage factors (CPF) as predicted from highly homologous proteins in S. cerevisiae. To determine whether polyadenylation and cleavage contributes to heterochromatin structure at the centromere repeats we focused on the function of Iss1, a subunit of CPF. We generated a C-terminal truncation of Iss1 (Iss1-C) removing 38 amino acids that, based on the Hermes insertions, were not important for viability. Iss1-C showed no growth restriction on non-selective medium but exhibited a heterochromatin defect as demonstrated by growth in the absence of uracil and reduced levels of H3K9 dimethylation (H3K9me2) at otr1R::ura4. These results demonstrate the Hermes screen correctly identified Iss1 as important for heterochromatin structure at the otr1R::ura4 reporter. Interestingly, we found Iss1 contributes to heterochromatin of centromere repeats in cells that lack the otr1R::ura4 reporter but in this case the contribution to H3K9me2 was only observed when the RNAi pathway was disabled by deletion of ago1. This role at the outer centromere repeats is therefore independent or redundant with RNAi. We expanded our study of the Iss1-C mutation to evaluate changes in expression and transcription termination genome-wide. RNA-seq data revealed Iss1-C did not significantly impact canonical transcription termination but 73 genes were found to have higher expression. Importantly, these genes overlapped significantly with genes upregulated in cells lacking Rrp6, the 3-5 exonuclease subunit of the nuclear exosome. Rrp6, as a key subunit of the nuclear exosome plays an important role in RNA surveillance in the degradation of meiotic transcripts expressed during vegetative growth and the resulting formation of heterochromatin at these genes. The elimination of meiotic mRNAs depends on the RNA binding protein Mmi1 to bind the determinant of selective removal (DSR) sequence in order to recruit the exosome. Our co-IP experiments revealed Iss1 interacted with Rrp6, Mmi1, and the polyA polymerase Pla1, indicating Iss1 is associated with this network of elimination factors. Significantly, this interaction with Mmi1 was disrupted by the Iss1-C mutation. We tested whether Iss1 plays a direct role in the heterochromatin of meiotic genes by performing ChIP-seq of Iss1-FLAG. While a subset of Iss1-bound genes was highly-expressed and was associated with the canonical function of Iss1 in mRNA termination, most Iss1-bound peaks showed a strong correlation with genes regulated by RNA elimination and heterochromatin. Importantly, the iss1-C mutation caused significant increases in RNA levels of these genes. Taken together, these studies of RNA levels, Iss1 association with chromatin, and H3K9me2 indicate that Iss1 plays a direct role in the formation of heterochromatin at meiotic genes. Our application of Hermes profiles to identify genes important for heterochromatin formation demonstrates the significance of the approach especially given that we were able to identify large numbers of essential genes, a result that is not obtainable with other screens.

在真核生物中，将DNA组装到高度凝结的异染色质中对于与基因组完整性相关的广泛功能至关重要。赖氨酸9（H3K9ME）上组蛋白H3的甲基化是通过为一系列对沉默的染色质蛋白创建结合位点来形成异染色质的核心。 S. pombe是一种出色的模型，已广泛用于研究产生和调节异染色质的分子机制。中心粒，亚近样组和交配型区域被包装到组成型异染色质中，而减数分裂基因被兼性异染色质沉默，直到细胞饥饿的氮。重要的是，CLR4，组蛋白H3K9甲基转移酶是通过多种机制募集到异染色质区域的。组成型异染色质来自RNAi因子，其中包括含有RNA诱导的转录沉默复合物（RITS）的AGO1。减数分裂基因的兼性异染色质与RNAi无关，并且依赖RNA消除因子RED1，MMI1和核外泌体。但是，在理解不同的处理活动如何靶向异染色质的过程中存在差距。识别基因功能的一种新方法是集成谱的高吞吐量测序，也称为TN-Seq，它识别了在选择性条件下生长重要的基因。在特定条件下维持增长所需的基因不能忍受在这种情况下的插入。 TN-Seq已应用于鉴定细菌中的致病基因。但是，我们开发了这种方法来鉴定酵母中的基本基因，随后其他人应用了该策略。 为了确定对异染色质重要的新因素的目标，我们使用爱马仕（Hermes）可替代元素和沉默报告基因（URA4）产生了密集的集成概况，该材料位于丝粒的外重复中。插入中断基因的插入，这些基因破坏了对异染色质激活的URA4重要的基因，因此无法在5-fuoror中生长出5 f fluoror的细胞。与缺乏记者的细胞相对于缺乏记者的细胞，在异染色质组装中既有作用的基因在细胞中的插入量明显较少。候选者的清单总共包含199个基因，重要的是，已知有65个对于生存能力至关重要。 这些必需基因是候选者，因为它们在3个序列中耐受了许多插入，这些插入降低了异染色质但不是生存力。大量必不可少的基因很重要，因为大多数发现对异染色质很重要的蛋白质都在无法包括必需基因的缺失菌株的筛选中鉴定出来。 199个候选人显示出对丝粒外重复序列沉默的功能的高度富集，并包括产生siRNA的所有四个因素。 鉴定出其他与异染色质结构无关的RNA加工因子。令人惊讶的是，四个RNA加工候选构成了典型mRNA聚腺苷酸化和切割因子（CPF）的相互作用模块，如酿酒酵母中高度同源蛋白所预测的。 为了确定聚腺苷酸化和裂解是否促进了Centromere重复的异染色质结构，我们集中在ISS1的功能上，ISS1的功能（CPF的亚基）。我们产生了ISS1（ISS1-C）的C末端截断，去除了38个氨基酸，该氨基酸基于爱马仕插入，对生存能力并不重要。 ISS1-C对非选择性培养基没有生长限制，但在没有尿嘧啶的情况下生长表现出异染色质缺陷，而OTR1R :: ura4处的H3K9二甲基化水平（H3K9ME2）降低。 这些结果表明，在OTR1R :: URA4 Reporter上，正确识别的HERMES屏幕对异染色质结构很重要。有趣的是，我们发现ISS1在缺乏OTR1R :: URA4报告基因的细胞中有助于Centromere重复序列的异染色质，但是在这种情况下，仅当RNAi途径因ago1的删除而禁用RNAi途径时，才观察到对H3K9me2的贡献。因此，在RNAi上，该作用在外侧粒粒重复序列是独立的或冗余的。 我们扩展了对ISS1-C突变的研究，以评估全面的表达和转录终止基因组的变化。 RNA-seq数据显示ISS1-C并未显着影响规范转录终止，但发现73个基因具有更高的表达。 重要的是，这些基因与缺乏RRP6的细胞（核外泌体的3-5核酸酶亚基）的细胞中上调的基因显着重叠。作为核外泌体的关键亚基RRP6在RNA监测中起着重要作用，在营养生长过程中表达的减数分裂转录物以及这些基因上异染色质的形成。 减数分裂mRNA的消除取决于RNA结合蛋白MMI1，以结合选择性去除（DSR）序列的决定因素以募集外泌体。我们的CO-IP实验表明，ISS1与RRP6，MMI1和Polya聚合酶PLA1相互作用，表明ISS1与该消除因子网络有关。 值得注意的是，这种与MMI1的相互作用被ISS1-C突变破坏了。 我们测试了ISS1是否通过执行ISS1-FLAG的chip-seq在减数分裂基因的异染色质中起着直接作用。尽管ISS1结合基因的子集高表达，并且与ISS1在mRNA终止中的规范功能有关，但大多数ISS1结合的峰显示与受RNA消除和异染色质调节的基因有很强的相关性。 重要的是，ISS1-C突变导致这些基因的RNA水平显着升高。综上所述，这些对RNA水平，ISS1与染色质和H3K9ME2的关联的研究表明，ISS1在减数分裂基因的异染色质形成中起着直接的作用。 我们对HERMES概况识别对异染色质形成重要的基因的应用证明了该方法的重要性，特别是考虑到我们能够识别大量的必需基因，这是其他筛查无法获得的结果。