Mechanisms of Transcriptome Aging Figure 2. Signature transcripts of CD4+ T-cells exhibit sex-specific shifts in gene expression with age in human skin. We identified 500 probe sets (from the Affymetrix Human Genome U133 Plus 2.0 array) representing signature transcripts that exhibit high expression in CD4+ T-cells isolated from peripheral blood (based on data obtained under GEO series accession GSE14278). Scatterplots (A) and (C) display expression of these transcripts in skin samples from young and old human subjects. Red symbols represent probe sets with higher expression in old subjects, and green symbols represent probe sets with higher expression in young subjects. The relative proportion of these two probe set groups is indicated by the pie chart shown in each figure. In (A) (females), young subjects were between 18 and 22 years of age (n = 5) and old subjects were between 57 and 75 years of age (n = 5). In (C) (males), young subjects were between 18 and 27 years of age (n = 5) and old subjects were between 57 and 60 years of age (n = 5). Among all T-cell-associated probe sets, the average increase in old subjects was 11% in females, but in males, T-cell-associated probe sets decreased by 5.3% on average (P,0.001; two-sample t-test). Across all probe sets, moreover, fold-change differences between old and young mice were negatively correlated between the sexes (r = 20.13, P,0.001). In parts (B) and (D), subjects were divided into different age groups (n = 2–3 subjects per group). 40-year fold-change estimates (old/young) were then averaged across subjects belonging to the same age group for each of the 500 probe sets associated with the CD4+ T-cell signature. Grey boxes denote the median and inter-quartile range for the 500 fold-change estimates in each age group. The red background region denotes an average fold-change larger than one (i.e., signature transcripts are age-increased on average), while the green background region denotes an average fold-change less than one (i.e., signature transcripts are age-decreased on average). doi:10.1371/journal.pone.0033204.g002 (Figures S3 and S4). Two binding site motifs (Foxl1 and Cebpa) were associated with age-increased expression in both sexes (Figures S3A, S3C, S4A and S4C). However, among top-ranked motif-expression associations, these were the only patterns consistent between sexes (Figures S3 and S4). One top-scoring motif – for the TF Nfe2l1 – was associated with age-decreased expression in females but in contrast was associated with age- increased expression in males (see magenta labels in left margin of Figures S3B, S3C, S4B and S4C). Thus, sex-dependence was noted with respect to both inflammatory and putative cis- regulatory mechanisms in humans (Figures 1, S3 and S4). We further investigated the associations between gene expres- sion and motif density observed for putative Zeb1 (zinc finger E- box binding homeobox 1), AP-2 (transcription factor AP-2 alpha) and YY1 (Yin and Yang 1) DNA binding site motifs (Figures S3B and S4B). In each case, a larger number of motif occurrences near the transcription start site (2000 BP upstream to 200 BP downstream) was associated with decreased expression in aging female skin, but trends were attenuated or contrasting in male skin (Figure S5). These female-specific motif associations were further supported based upon analysis of intergenic sequences, intronic sequences and TSS-proximal sequences conserved among verte- brate species (see Methods; results for AP-2 are shown in Figure S6). With respect to intronic regions, associations between Zeb1, AP-2 and YY1 motifs and age-related expression diverged sharply between sexes, with significant but opposite patterns in females relative to males (Figures S6E–S6H). PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33204