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The broad impact of environmental exposures on repetitive element expression in cellular biology

环境暴露对细胞生物学中重复元件表达的广泛影响

基本信息

批准号：
10252594
负责人：
Richard Woychik
金额：
$ 12.49万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
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/10252594
关键词：
1 year old Alleles Animals Antibodies Appearance Area Back Biogenesis Biological Brain Brain region Cells Cellular biology Cerebellum Chimeric Proteins Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Code Color DNA DNA Methylation Data Set Defect Diabetes Mellitus Diet Ectopic Expression Elements Embryo Environmental Exposure Environmental Impact Epigenetic Process Estrogen Receptors Evaluation Exons Exposure to Expression Profiling Female Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Goals Housing Link Maize Metabolic MicroRNAs Mitochondria Modification Motor Mus Mutagens Mutant Strains Mice Mutation Neurons Nuclear Nuclear Receptors Obesity Oligodendroglia Organism Outcome Oxygen PPARG gene Pathologic Penetrance Peroxisome Proliferators Phenotype Play Post-Translational Protein Processing Production Protein Isoforms Proteins RNA RNA Splicing Repetitive Sequence Research Role Rotarod Performance Test Sampling Series Short Interspersed Nucleotide Elements Short Tandem Repeat System Tail Testing Time Transcript Transcription Coactivator Translating Untranslated RNA Up-Regulation Variant Water Western Blotting Work aged agouti protein base behavior test cell type drug of abuse environmental agent epigenetic regulation genome-wide histone modification insight interest mitochondrial metabolism mutant programs promoter response ribosome profiling sexual dimorphism transcriptome sequencing whole genome

项目摘要

The purpose of this project is to determine the role that repetitive elements (REs) play in the biological outcome of environmental exposures. While it is known that the expression of REs changes in response to environmental agents, mechanistic insights into the impact of REs on the biology of cells and organisms is an area of research that has not been explored in depth. We are specifically interested in studying the extent to which REs alter the expression of adjacent genes through the formation of fusion transcripts (FTs). We chose to use RNA-seq to study this problem, and we have developed a robust analytical pipeline to detect FTs. In our initial study, we have found that many genes in the brain of mice are expressed as FTs, including the peroxisome proliferator gamma co-activator 1 (Pgc1). Pgc1 is a master metabolic regulator that was identified back in 2004 as a co-transcriptional activator of the mitochondrial biogenesis program. Our analytical strategy identified two repeat-containing isoforms of Pgc1 in the mouse brain: one involving a simple sequence repeat (SSR), about 500 Kb upstream from the canonical promoter that spliced to the second coding exon of the gene. The second fusion isoform involved the same SSR that spliced to a SINE (small interspersed nuclear element), that is about 250 Kb downstream from it, and then spliced to the second coding exon. Analysis of limited publicly available RNA-seq data sets revealed that both of these new FT isoforms are brain specific. Moreover, within the brain, the SSR-SINE-exon 2 isoform seems to be confined to neurons, while we detected the SSR-exon 2 isoform only in oligodendrocytes. We also analyzed publicly available ribosomal profiling data sets and found evidence that the SSR-containing isoforms are actively translated in the brain. Additional support that these new FTs make proteins come from our work in which the cloned SSR-SINE-exon 2 isoform containing a myc tag were found to give rise to proteins of the expected size on a western blot. We also have generated antibodies to the amino-termini of these fusion proteins as well as the wild-type. Our initial analysis demonstrated that the antibodies that recognize the C-terminus, or the second coding exon, are very specific. We are still working on optimization of the work with the antibodies, but we have been able to demonstrate that an antibody to the protein predicted to be expressed from the SSR-SINE-exon2 FT does not produce a protein in homozygous mutant animals that we generated using the CRISPR-cas system (see below). We continued to characterize the mouse that carries a 4 bp deletion that is just downstream from the predicted ATG in the SINE element. Detailed analysis of the brains of the mutant mice revealed no obvious pathological defects and the animals do not show any specific visible phenotypes over time (animals are now about 1 year old) under normal housing conditions. We have performed a series of behavioral tests in the animals, including the water maize, open field and rotarod with young and aged animals. While no changes were observed in most tests, the mutant animals show a dramatic phenotype associated with the rotarod test that is most notably observed only in females. We had initially evaluated gene expression profiles in the cerebellum of the mutant mice, since this region of the brain has been linked to motor and coordination defects that are detectable with the rotarod test. Because we had unexpectedly found substantial up-regulation of a number of genes in the mutant animals, we replicated the microarrays with several independently prepared RNA samples from independent animals. In the past year we have focused on trying to understand the differences in gene expression, particularly why they occurred more prominently in females. Our recent analysis has indicated that it is likely that this new isoform of PGC1a interacts in neurons with the estrogen receptor and/or with PPARG, a nuclear receptor that has been shown to cause sexual-dimorphic gene expression programs.

该项目的目的是确定重复元素（RE）在环境暴露的生物结果中所起的作用。虽然已知RE的表达响应于环境因子而变化，但RE对细胞和生物体生物学的影响的机制见解是尚未深入探索的研究领域。我们特别感兴趣的是研究RE通过形成融合转录本（FT）改变相邻基因表达的程度。我们选择使用RNA-seq来研究这个问题，我们已经开发了一个强大的分析管道来检测FT。在我们最初的研究中，我们发现小鼠大脑中的许多基因表达为FT，包括过氧化物酶体增殖物γ共激活因子1（Pgc 1）。pgc 1是一种主要的代谢调节因子，早在2004年就被鉴定为线粒体生物发生程序的共转录激活因子。我们的分析策略确定了两个重复含有异构体的Pgc 1在小鼠大脑中：一个涉及一个简单的序列重复（SSR），约500 Kb上游的典型启动子，剪接到第二个编码外显子的基因。第二种融合同种型涉及与SINE（小散布核元件）剪接相同的SSR，SINE（小散布核元件）位于其下游约250 Kb处，然后剪接至第二编码外显子。对有限的公开可用的RNA-seq数据集的分析显示，这两种新的FT亚型都是脑特异性的。此外，在脑内，SSR-SINE-外显子2亚型似乎仅限于神经元，而我们仅在少突胶质细胞中检测到SSR-外显子2亚型。我们还分析了公开的核糖体分析数据集，并发现证据表明，含有SSR的亚型在大脑中被积极翻译。这些新的FT产生蛋白质的额外支持来自我们的工作，其中发现克隆的含有myc标签的SSR-SINE-外显子2同种型在蛋白质印迹上产生预期大小的蛋白质。我们还产生了针对这些融合蛋白以及野生型的氨基末端的抗体。我们的初步分析表明，识别C-末端或第二编码外显子的抗体是非常特异的。我们仍在努力优化抗体的工作，但我们已经能够证明，预测从SSR-SINE-exon 2 FT表达的蛋白质的抗体不会在我们使用CRISPR-cas系统产生的纯合突变动物中产生蛋白质（见下文）。我们继续表征携带4 bp缺失的小鼠，该缺失恰好位于SINE元件中预测的ATG的下游。对突变小鼠大脑的详细分析显示，在正常饲养条件下，没有明显的病理学缺陷，并且随着时间的推移，动物没有显示出任何特定的可见表型（动物现在约1岁）。本研究对幼龄和老龄动物进行了一系列行为学试验，包括水玉米、旷场和转棒。虽然在大多数试验中未观察到变化，但突变动物显示出与旋转杆试验相关的显著表型，仅在雌性动物中观察到最显著的表型。我们最初评估了突变小鼠小脑中的基因表达谱，因为大脑的这一区域与旋转杆测试可检测到的运动和协调缺陷有关。因为我们意外地发现了突变动物中大量基因的上调，我们用几个独立制备的来自独立动物的RNA样本复制了微阵列。在过去的一年里，我们一直致力于了解基因表达的差异，特别是为什么它们在女性中更突出。我们最近的分析表明，这是可能的，这种新的异构体PGC 1a与雌激素受体和/或与PPARG，核受体，已被证明会导致性二态基因表达程序在神经元中相互作用。