Discriminating between causes of age-related variegated gene expression

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，这种细胞间的变异 二是随着年龄的增长而增加。组织中的细胞需要协调基因表达以维持稳态。 3因此，失调的变异可能与生理能力随年龄的丧失有关，或者是其原因。 然而，这种增加的基因表达变异的原因和后果尚不清楚。在这 5项目我建议使用C.线虫和人类组织培养作为研究年龄相关变异的模型系统 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， 7种报告基因在幼龄C. elegans动物;也就是说，对于许多基因， 在幼年动物中，基因A与基因B的表达比例在X型细胞中是固定的，并且与在细胞中不同。 Y型9例。我们发现，这种固定的表达模式随着年龄的增长而恶化;在给定的细胞类型中， 10在个体动物中以可变化学计量表达。类似地，在哺乳动物中， 在老化或衰老期间观察到的11表达也是不相关的。因此，我建议， 12同源细胞随年龄的差异性可能是衰老的保守现象。我指的是增加 13个与年龄无关的基因表达变异为年龄相关的杂色基因表达（VGE）。在 14提出的项目，我将利用这两个权力的C。线虫和人类细胞培养来研究 随着年龄的增长，同源细胞变得越来越不相似。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 17 K99和R 00，我将调查导致年龄相关VGE的潜在原因。K99-Aim 1：我会的 18确定等位基因接近随年龄的变化是否有助于C. elegans通过分析表达， 19个相同的启动子整合在姐妹染色体上的相同基因座上。K99-Aim 2：我会学习和使用 20个单细胞RNA-seq，以确定等位基因偏倚/单等位基因现象在人成纤维细胞中的患病率是否随年龄增加而增加。 21 R 00-Aim 3：我将确定哪些基因和途径随着年龄的增长而高度表达。我会 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是随机的或适应性的。 这些基因的23个报告者预测压力抵抗力，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 R 00-目的4：使用人类细胞培养，我将确定细胞间的通讯是否会在人类细胞中传播VGE。 26个具有年轻表达模式的细胞-即没有VGE的细胞。延期更新：延期期间 27期的颁奖，我将集中精力完成目标2。在最初的支持期间，我 28优化了感兴趣的转录本的富集，并在分析 使用单细胞RNA-seq.利用这一技术进步，我将能够完成 衰老人细胞中等位基因表达变异的分析。

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.