Discriminating between causes of age-related variegated gene expression

项目总结： 同一组织中的细胞可以在不同水平上表达完全相同的基因，这种细胞间的差异 往往随着年龄的增长而增加。组织中的细胞需要协调基因的表达来维持动态平衡。 因此，失调的变异可能与随着年龄增长而丧失生理能力有关，也可能是导致这种情况的原因。 然而，这种基因表达差异增加的原因和后果尚不清楚。在这 项目一建议使用线虫和人体组织培养作为模型系统来研究与年龄相关的变异。 在受控环境中同源细胞之间的基因表达。在我们之前的研究中，我们发现， 许多报告基因在幼年线虫动物中的细胞特异性表达模式；即许多基因 在幼年动物中，基因A和基因B的表达比例在X细胞中是固定的，与细胞中的不同 我们发现，这种固定的表达模式会随着年龄的增长而恶化；在给定的细胞类型中，基因会变成 在个体动物中表现为不同的化学计量比。同样，在哺乳动物中，基因的细胞间变异 在衰老或衰老过程中观察到的表达也是无关的。因此，我建议不断增长 同源细胞与年龄的不同可能是一种保守的衰老现象。我指的是增长 无关基因表达随年龄的变化称为年龄相关基因表达(VGE)。在 提议的项目，我将利用线虫的力量和人类细胞培养来研究 同源细胞随着年龄的增长变得更加不同。在项目的K99阶段，我将学习以下技术 定量显微镜、单细胞RNAseq和关注衰老的人类细胞培养方法。在整个过程中 K99和R00，我将调查导致年龄相关VGE的潜在原因。K99-Aim1：我会 基因表达分析确定等位基因获取随年龄的变化是否对线虫的VGE有贡献 在姐妹染色体上的相同位置整合的相同启动子。K99-AIM2：我会学习和使用 单细胞RNA-SEQ以确定人类成纤维细胞中等位基因偏向/单等位基因的流行率是否随着年龄的增长而上升。 R00-Aim3：我将确定哪些基因和途径会随着年龄的增长而高度可变地表达。这就做 通过确定这些基因的表达水平来检查这些基因的可变表达是否是随机的或适应性的 这些基因的记者可以预测抗压性、健康或寿命。这些实验将解决 假设单个细胞对年龄相关压力的适应性生理反应有助于VGE。 R00-Aim 4：使用人类细胞培养，我将确定细胞间的通讯是否在 具有年轻表达模式的细胞--即没有VGE的细胞。

Project Summary: Cells in the same tissue can express the exact same gene at different levels, and this cell-to-cell variation tends to increase with age. Cells in tissues need to coordinate gene expression to maintain homeostasis. Thus, dysregulated variation may be related to, or causative of, the loss of physiological capacities with age. However, the causes and consequences of this increased gene expression variation are not known. In this project I propose to use C. elegans and human tissue culture as model systems to study age-related variation in gene expression between homologous cells in controlled environments. In our prior study, we found strong, cell-specific expression patterns for many reporter genes in young C. elegans animals; that is, for many genes in young animals, the ratio of gene A to gene B expression was fixed in cell type X, and different than in cell type Y. We found that this fixed expression pattern deteriorates with age; in a given cell type genes become expressed at variable stoichiometry in individual animals. Similarly, in mammals, cell-to-cell variation in gene expression observed during aging or senescence is also uncorrelated. Thus, I propose that growing dissimilarity between homologous cells with age may be a conserved phenomenon of aging. I refer to increase of uncorrelated gene expression variation with age as age-related variegated gene expression (VGE). In the proposed project, I will harness both the power of C. elegans and human cell culture to investigate how homologous cells become more dissimilar with age. In the K99 phase of the project I will learn techniques for quantitative microscopy, single cell RNAseq and aging-focused human cell culture methods. Throughout the K99 and R00, I will be investigating potential causes that contribute to age-related VGE. K99-Aim1: I will determine if changes of allele access with age contribute into VGE in C. elegans by analyzing expression from of identical promoters integrated at the identical loci on sister chromosomes. K99-Aim2: I will learn and use single-cell RNA-seq to determine if prevalence of allele bias/monoallelism rises in human fibroblasts with age. R00-Aim3: I will determine what genes and pathways become highly variably expressed with age. I will examine if variable expression of these genes is stochastic or adaptive by determining if expression levels of reporters of these genes predict stress resistance, health or lifespan. These experiments will address the hypothesis that adaptive physiological responses of individual cells to age-related stress contribute to VGE. R00-Aim 4: Using human cell culture, I will determine if cell-to-cell communications propagate VGE among cells with youthful expression patterns – that is, cells without VGE.