Lack of global correspondence between aging effects in human sun-protected skin and tail skin from CB6F1 mice in both sexes A previous investigation of aging in brain, muscle and kidney found limited correspondence between age-related expression profiles in humans and inbred C57BL/6 mice [24]. We thus evaluated gene expression in tail skin of young (5 month) and old (30 month) CB6F1 hybrid mice of both sexes (n = 5 per age-sex combination; Affymetrix Mouse Genome 430 2.0 Array platform). Inspection of aging signatures, including mouse and human skin combined with other mammalian tissues, highlighted a general pattern by which aging patterns in human tissues clustered together with each other and apart from those of mouse tissues (Figure 3). The human and mouse fold-change estimates (old/ young) were negatively correlated or uncorrelated (Pearson r = 20.096 in females, P,0.001; r = 20.002 in males, P = 0.77; see Figure 4). This lack of significant similarity was confirmed based upon three other analytical methods, including examination of differential expression signature overlap [11] (Figure S7), rank- based AUC statistics [35] (P$0.136; Figure S8) and gene set enrichment analysis [36] (P$0.51; Figure S9). Global effects of aging on gene expression in CB6F1 mouse tail skin therefore do not have significant similarity to those in sun-protected human skin. Aging does not increase expression of Lyz1, Lyz2, Clec7a, Cd48, Tyrobp and Mpeg1 in tail skin of male and female CB6F1 mice A robust and striking effect of aging in laboratory mice is elevated expression of genes associated with immune activation, including cathepsin S (Ctss), complement components (e.g., C1qa), cluster of differentiation antigens (Cd48) and lysozyme (Lyz1 and Lyz2) (see Figure 6 from [11]). We compared genes regulated by aging in mouse tail skin with age-regulated genes identified in 28 other datasets with young and old samples from a diverse panel of mouse tissues (e.g., lung, liver, kidney, spleen, aorta, muscle, heart, cochlea, eye and central nervous system; all studies used the same Affymetrix Mouse Genome 430 2.0 platform; total of 295 arrays; Figures 5 and S10; Table S2). As expected, the most consistent effect of aging in mouse tissues was elevated expression of membrane-associated immune response genes, including glycoprotein 49 A (Gp49a), lysosomal-associated protein transmembrane 5 (Laptm5), CD52 antigen (Cd52), CD53 antigen (Cd53), C-type lectin domain family 7 member a (Clec7a), lysozyme 1 (Lyz1) and lysozyme 2 (Lyz2) (see Figure 5). Strikingly, however, in mouse tail skin, expression of these genes was decreased, in both sexes, but particularly in females (Figure 5). This aberrant pattern was further illustrated by inspection of genes associated with specific immune-associated GO terms (e.g., activation of immune response, positive regulation of defense response, acute inflammatory response), which were decreased with age in CB6F1 tail skin, but increased in most other mouse tissues (see Figure S11B). We confirmed by quantitative RT- PCR that expression of Clec7a, Lyz1 and Lyz2 was decreased with age in mouse tail skin (Figures 6, S12 and S13). Further evaluation indicated that the pattern was specific to tail skin and not, for instance, an unusual aging effect in CB6F1 mice. In particular, one or more of the evaluated genes (Clec7a, Lyz1 and Lyz2) was significantly elevated with age in ear skin, heart, lung, kidney and small intestine of CB6F1 mice (Figures 6, S12 and S13). Mechanisms of Transcriptome Aging Aging of tail skin in CB6F1 mice promotes decreased expression of transcripts associated with antigen presenting cells in both sexes C-type lectin domain family 7 (Clec7a/Dectin-1) was unexpect- edly decreased by aging in mouse tail skin (Figures 5 and 6) and is expressed at high levels in leukocyte subsets, including dendritic cells, macrophages, monocytes and neutrophils [37]. We thus hypothesized that mouse tail skin would exhibit concerted declines in the expression of transcripts associated with specific lymphocyte subsets. Consistent with this, inflammation profiles indicated decreased expression of transcripts associated with antigen presenting cells (APC; DC and macrophages) in old mouse tail skin as compared to young skin (both sexes), along with weak elevation of transcripts associated with CD4+ T-cells (Figures 7 and S14). This decline contrasted with a general trend observed across mouse tissues, in which APC-associated transcripts were frequently elevated with age (Figure 7). Moreover, although aging decreased expression of genes expressed in the T- and B-cell subsets in human males, an age-related decrease in the expression of transcripts associated with APC was not noted in either female or male human skin (Figure 1). Thus, differential age-related declines in DC and macrophage-associated expression contribute to divergent effects of transcriptome aging in human skin and CB6F1 tail skin (Figures 3 and 4). Zeb1 and Prrx2 DNA binding site motifs are associated with gene expression changes with aging of CB6F1 mouse tail skin We screened 541 mammalian transcription factor DNA binding site motifs for association with gene expression changes in tail skin from aging mice (Figures 8 and S15). As compared to humans (Figure S3 and S4), consistent trends between species included age- increased expression of genes associated with elevated myeloid zinc finger 1 (Mzf1) and SRY-box 10 (Sox10) binding site density in males (Figures 8C and S15C; blue labels). Notably, however, we identified five motifs associated with age-increased expression in tail skin from female CB6F1 mice, but age-decreased expression in human females (i.e., AP-2, Myb, Myc, Nr4a2 and Zeb1; Figures 8A and S15A), along with two motifs associated with age-decreased expression in female mice but age-increased expres- sion in human females (i.e., Nkx2 and Nobox; Figures 8B and S15B). Moreover, in males, two motifs were linked with age- decreased expression in mice but age-increased expression in humans (i.e., Foxl1 and Gata2; Figures 8D and S15D). Thus, in line with the lack of correspondence between age-associated gene expression patterns in humans and mice (Figures 3 and 4), as well as disparate shifts in inflammatory gene expression patterns with aging in each system (Figures 1 and 7), we identified contrasting motif-expression associations with aging of human and mouse skin. In mice, motifs strongly associated with tail skin aging included Zeb1 (zinc finger E-box binding homeobox 1) and Prrx2 (paired related homeobox 2) (Figures 8 and S15). These motif-expression associations were consistent between mouse sexes, with the Zeb1 motif linked to increased expression with age, and the Prrx2 motif linked to decreased expression with age (Figure S16). In each case, moreover, a similar motif-expression association was discernible in intergenic and intronic regions of mouse genes, as well as in TSS- proximal sequences conserved among vertebrate species (see Methods; results for Zeb1 are shown in Figure S17). PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33204