Discriminating between causes of age-related variegated gene expression

同一组织中的细胞可以在不同水平表达完全相同的基因，这种细胞间的差异 2有随年龄增长的趋势。组织中的细胞需要协调基因的表达来维持动态平衡。 3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，这种基因表达变异增加的原因和后果尚不清楚。在这 项目一建议以线虫和人体组织培养为模型系统，研究与年龄相关的变异。 6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， 在幼年线虫动物中许多报告基因的7种细胞特异性表达模式；即许多基因 8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 10在个体动物中以不同的化学计量比表达。同样，在哺乳动物中，基因的细胞间变异 11在衰老或衰老过程中的表达也是不相关的。因此，我建议不断增长 12同源细胞随年龄的不同可能是一种保守的衰老现象。我指的是增长 13个与年龄无关的基因表达随年龄变化为年龄相关基因表达(VGE)。在 14提议的项目，我将利用线虫的力量和人类细胞培养来研究如何 15个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 16定量显微镜、单细胞RNA-SEQ和关注衰老的人类细胞培养方法。在整个过程中 17 K99和R00，我将调查导致年龄相关VGE的潜在原因。K99-Aim1：我会 18通过分析以下基因的表达，确定等位基因访问随年龄的变化是否对线虫的VGE有贡献 19个相同的启动子整合在姐妹染色体上的相同座位上。K99-AIM2：我会学习和使用 20单细胞RNA-SEQ以确定人类成纤维细胞中的等位基因偏向/单等位基因是否随着年龄的增长而增加。 21 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 22通过确定这些基因的表达水平，检查这些基因的可变表达是否具有随机性或适应性 这些基因的23名记者预测了抗压性、健康或寿命。这些实验将解决 24假设个体细胞对年龄相关应激的适应性生理反应有助于VGE。 25 R00-Aim 4：使用人类细胞培养，我将确定细胞之间的通信是否在 26个具有年轻表达模式的细胞--即没有VGE的细胞。扩展更新：在扩展期间 在27期的奖励期间，我将专注于完成目标2。在最初的支持期间，我已经 28优化了对感兴趣的转录的丰富，并在分析方面取得了重大的方法学进展 29等位基因的表达采用单细胞RNA-seq。利用这一技术进步，我将能够完成 30衰老人体细胞等位基因表达变异分析。

1 Cells in the same tissue can express the exact same gene at different levels, and this cell-to-cell variation 2 tends to increase with age. Cells in tissues need to coordinate gene expression to maintain homeostasis. 3 Thus, dysregulated variation may be related to, or causative of, the loss of physiological capacities with age. 4 However, the causes and consequences of this increased gene expression variation are not known. In this 5 project I propose to use C. elegans and human tissue culture as model systems to study age-related variation 6 in gene expression between homologous cells in controlled environments. In our prior study, we found strong, 7 cell-specific expression patterns for many reporter genes in young C. elegans animals; that is, for many genes 8 in young animals, the ratio of gene A to gene B expression was fixed in cell type X, and different than in cell 9 type Y. We found that this fixed expression pattern deteriorates with age; in a given cell type genes become 10 expressed at variable stoichiometry in individual animals. Similarly, in mammals, cell-to-cell variation in gene 11 expression observed during aging or senescence is also uncorrelated. Thus, I propose that growing 12 dissimilarity between homologous cells with age may be a conserved phenomenon of aging. I refer to increase 13 of uncorrelated gene expression variation with age as age-related variegated gene expression (VGE). In the 14 proposed project, I will harness both the power of C. elegans and human cell culture to investigate how 15 homologous cells become more dissimilar with age. In the K99 phase of the project I will learn techniques for 16 quantitative microscopy, single-cell RNA-seq and aging-focused human cell culture methods. Throughout the 17 K99 and R00, I will be investigating potential causes that contribute to age-related VGE. K99-Aim1: I will 18 determine if changes of allele access with age contribute into VGE in C. elegans by analyzing expression from 19 of identical promoters integrated at the identical loci on sister chromosomes. K99-Aim2: I will learn and use 20 single-cell RNA-seq to determine if prevalence of allele bias/monoallelism rises in human fibroblasts with age. 21 R00-Aim3: I will determine what genes and pathways become highly variably expressed with age. I will 22 examine if variable expression of these genes is stochastic or adaptive by determining if expression levels of 23 reporters of these genes predict stress resistance, health or lifespan. These experiments will address the 24 hypothesis that adaptive physiological responses of individual cells to age-related stress contribute to VGE. 25 R00-Aim 4: Using human cell culture, I will determine if cell-to-cell communications propagate VGE among 26 cells with youthful expression patterns – that is, cells without VGE. Extension update: During the extended 27 period of the award, I will focus on completion of the Aim 2. During the initial period of the support, I have 28 optimized enrichment of transcripts of interest and achieved significant methodological progress in analysis of 29 allele expression using single-cell RNA-seq. Using this technical progress, I will be able to complete the 30 analysis of allelic expression variation in senescent human cells.