Mechanisms of Transcriptome Aging Figure 4. Weak association between aging effects in sun-protected human skin and tail skin from CB6F1 mice. The expression fold- change (old/young) over 40 years (human) or 2 years (mouse) was calculated for 15,203 transcripts associated with orthologous human and mouse genes. The scatterplot shows the human-mouse association between fold change estimates in (A) female human subjects versus female mice and (B) male human subjects versus male mice. The dashed blue line represents the least squares regression estimate, and the estimated Pearson correlation (r) is shown in the lower right quadrant (blue font). The yellow region in (A) and (B) outlines the central 50% of data points closest to the bivariate median (based upon Mahalanobis distance). The grey region outlines the central 80% of data points closest to the bivariate median (based upon Mahalanobis distance). A bivariate association, if present, would be indicated by an oblong (non-circular) shape of the yellow or grey region. In (A), the estimated correlation coefficient is significantly negative (r = 20.096; P,0.001). In (B), the estimated correlation coefficient is non-significant (r = 20.002; P = 0.77). Absence of significant correspondence between human and mouse aging effects was further supported based upon three other statistical methods for comparing gene expression profiles (see Figures S7, S8 and S9). doi:10.1371/journal.pone.0033204.g004 Clusterin (Clu) expression is elevated with age in CB6F1 mice and decreased in long-lived Pit1(dw/dw) mice We found that aging has a unique effect in mouse tail skin, as compared to other tissues, with an unexpectedly weak inflamma- tory gene expression signature (Figures 5 and S11B). Nevertheless, we expected that some aspects of tail skin aging would be closely tied to basic aging mechanisms in other tissues, both in mice and humans. To identify such responses, we aligned expression signatures of aging from human and mouse skin (both sexes), in combination with signatures from a diverse panel of human and mouse tissues (Figure S18; Tables S1 and S2). Inspection of these meta-profiles uncovered expression responses to aging that were similar across many human and mouse tissues (e.g., Clu, Erbb2ip and C5ORF13/D0H4S114; see Figure S18). The protein-damage biomarker Clu, for instance, has been linked to Alzheimer’s disease in genetic association studies [38–41], and was previously identified as part of a common signature of genes with elevated expression in aging mammalian tissues [23]. In agreement, our meta-profiles indicated that Clu expression increased with age in 13 of 27 human aging profiles and 14 of 30 mouse aging profiles (Figures 9 and S18A). This is a better-supported aging effect than, for instance, elevated expression of Cdkn2a (p16INK4a) [42], which was increased by aging in 10 of 27 human profiles but was likewise elevated in only 4 of 30 mouse profiles (data not shown). Further evaluation by RT-PCR revealed that Clu is elevated with age in cardiac tissue (males) and kidney (females) of CB6F1 mice, but surprisingly, decreased with age in ear skin (Figures 9D– 9G). One application of well-supported gene expression biomark- ers of aging is to judge whether intrinsic aspects of the aging process have been influenced by genetic or dietary interventions (e.g., GH/IGF-1 mutations or calorie restriction). As a test of this concept, we evaluated expression of Clu in GH-deficient Pit1(dw/ dw) mice, which exhibit increased longevity and are viewed as a mouse model of healthy aging [43]. In both liver and hypothalamus, expression of Clu was diminished in Pit1(dw/dw) mice at 6 months of age, consistent with a ‘‘delayed aging’’ phenotype of Pit1(dw/dw) mice (P = 0.005; Figures 9H and 9I). Expression of Clu is thus sensitive to key endocrine pathways that modulate development of age-related pathology in mice. Discussion Senescent decline of skin function provides a model for understanding basic biological mechanisms of aging that operate in mammalian organ systems and cell types. We have used genome-wide expression analysis to identify intercellular (inflam- matory) and intracellular (cis-regulatory) mechanisms that account for the large-scale shifts in gene expression observed with intrinsic aging of human and mouse skin. In female subjects, inflammatory gene expression was heightened with age, with decreased expression of genes near regions of high Zeb1, AP-2 and YY-1 motif density. In male subjects, however, these effects were contrasting and absent, respectively. Such sex disparities were not observed in mouse tail skin, and at a broad level, there was little human-mouse correspondence between aging effects. Comparison of aging signatures across multiple datasets revealed that, surprisingly, aging in tail skin does not increase expression of immune response genes typically elevated with age (e.g., Clec7a, Lyz1, Lyz2, Cd48 and Tyrobp). We thus present a counter-example to what is the most robust expression signature of aging known to occur in mice. This unexpected result is consistent with a decline in the abundance of antigen presenting cells (APCs) with aging of tail skin in CB6F1 mice, and we propose that this is one factor that contributes to the observed disparity of aging effects between human sun-protected skin and mouse tail skin. Genome-wide expression profiling studies have repeatedly observed that transcripts elevated with age are linked to inflammation and immune response [11,23,44–47]. These expres- sion patterns are probable indicators of inflammation and infiltration of aging tissues by leukocyte populations [30]. In this study, we have generated in silico profiles that characterize such shifts in lymphocyte abundance with aging in both humans and PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33204