Mechanisms of Transcriptome Aging Figure 6. Clec7a expression is decreased with age in tail skin but increased by age in heart and lung from CB6F1 mice. RT-PCR was used to evaluate the expression of Clec7a in mice from three age groups (5 months, 17 months and 30 months) and both sexes (n = 5–6 mice for each sex/age combination). The expression of 18S ribosomal RNA (Rn18s) was used as an internal control gene for calculation of relative expression in each sample. Plots (A)–(F) show mean expression levels in each group (6 one standard error), which have been normalized to the average expression level of the young (5 month) female group. In each plot, and for each sex, the average expression of middle-aged (17 month) and old groups (30 months) was compared to that of the young group (5 months). A blue star is used to indicate a significant difference based upon a two-sample non- parametric statistical test (P,0.05; either Wilcoxon or Kruskal-Wallis rank sum test). doi:10.1371/journal.pone.0033204.g006 properties of female sex hormones, such as estrogen, contribute to this sex disparity in skin and the central nervous system [48–50]. In females, estrogen levels decline with age, but more precipitously following menopause, while in males, dominance of estrogen relative to testosterone increases with age in a more continuous fashion [51]. These temporal dynamics are consistent with the gene expression signature associated with CD4+ T-cells, which was age-independent in females prior to the age of 40, and subsequently exhibited its strongest elevation with respect to the early 40’s age group (approximately 40–45; see Figure 2B). In contrast, for male subjects, the CD4+ T-cell signature exhibited a slight and continuous decline with advancing age (Figure 2D). Previous work has identified transcription factors associated with skin aging, including ETS1, ETS2, AP-1, p53, E2F1 and NF- kB [8,19,27,28]. In this study, we identified motif-expression associations that include transcription factors not previously implicated in skin aging mechanisms (e.g., Zeb1, AP-2 and YY1 motifs in female human subjects, see Figures S3 and S4; Zeb1 and Prrx2 in female and male mice, see Figures 8 and S15). These results provide data-driven hypotheses for future work, although we expect that further experimental evaluation will be necessary to understand the potential regulatory role of these transcription factors in young versus old skin tissue. Importantly, however, our findings give emphasis to the sex- and species-specific character of these motif-expression associations, suggesting that sex and species must be carefully accounted for both in the analysis of genomic data and in mechanistic studies of such factors and their contribution to skin aging. The Zinc finger E-box binding homeobox 1 (Zeb1) transcription factor provides an illustrative example. This transcription factor was associated with age- PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33204