Discriminating between causes of age-related variegated gene expression

University Of Washington, Seattle WA

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Abstract

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.