A, B, C and D, respectively). Filled triangles indicate whether average fold-change estimates among these genes are significantly high (up-triangle; P,0.05) or significantly low (down-triangle; P,0.05). Triangles are displayed only if significant p-values were obtained from each of three separate tests evaluating whether the average fold-change estimate (or distribution of estimates) is significantly high or low (i.e., one-sample t-test, exact binomial proportion test and Wilcoxon signed rank test; triangles denote P,0.05 in each test). GO terms have been clustered based upon a similarity metric that is proportional to the number of shared ancestral terms in the GO hierarchy. (PDF) Figure S3 Sex-specific associations of transcription factor binding site motifs with the gene expression response to aging in human skin. The chart lists top-scoring motifs associated with genes (A) increased by aging in female subjects, (B) decreased by aging in female subjects and (C) increased by aging in male subjects. A total of 541 position weight matrices (PWMs) associated with mammalian transcription factors were obtained from the Jaspar and UniPROBE databases. For each PWM, regions proximal to the annotated transcription start site (TSS) of known human genes were scanned to identify putative binding sites (2000 BP upstream, 200 BP downstream). Based on results from this scan, we identified genes associated with varying numbers of binding sites (1…n) for a given motif. The figure provides a ‘‘threshold-free’’ display of trends relating binding site abundance to age-associated gene expression patterns. We derived a series of gene sets (x1, x2… xn), where the first set x1 contained genes with at least one binding site in the search region, set x2 contained genes with at least two binding sites, and so on, with set xn containing genes with at least n binding sites. The number of genes belonging to each set declined successively in the series spanning set x1 to xn. The number of sets evaluated in this fashion (n) varied among the 541 PWMs considered, since more complex PWM models had fewer genome matches within search regions. For each PWM and each gene set, the proportion of genes increased by aging in females (A and B) or males (C) was evaluated (horizontal axis). For sets x , x2,…, x , this proportion is 1 n represented by open circles, with blue circles representing sets for which the ratio of age-increased to age-decreased genes is significantly different from 0.50 (P,0.05; Fisher’s exact test), and yellow circles representing sets for which the ratio is significant at a more stringent threshold (FDR-adjusted P,0.05; Benjamini- Hochberg method). Circle size corresponds to the number of genes included in each set (see legend in right margin), where larger symbols denote larger sets of genes with fewer PWM motif matches, and smaller circles denote smaller sets of genes with more PWM motif matches. In parts (A)–(C), PWMs have been ranked according to the most extreme ratio (i.e., most different from 0.50) obtained for any set including at least 100 genes. For each age- associated motif, candidate target genes are listed in the right margin (red and green font). Candidate target genes were identified as those strongly altered by aging (P,0.05), with a maximal number of motif occurrences within the search region (age-increased genes in red font; age-decreased genes in green font). We noted that two top-scoring motifs (blue font, left margin) exhibited a similar relationship with aging in female versus male skin, while for one motif (magenta font, left margin) there was a contrasting relationship with aging in females versus males. Motifs associated with age-decreased expression in males are not shown because no gene sets with ratios (no. age-increased genes/no. age- decreased genes) significantly less than 0.50 were identified in males. (TIF) Mechanisms of Transcriptome Aging Figure S4 Sex-specific associations of transcription factor binding site motifs with the gene expression response to aging in human skin. This figure is identical to Figure S3, except a simulation-based correction for multiple testing has been applied (yellow symbols). For each simulation trial, genes were randomly assigned to gene sets (631 sets in total), where the respective size of gene sets was matched to those evaluated in our analysis. Following this random assignment of genes to gene sets, p-values were generated from each set to test for age-biased expression (Fisher’s Exact Test), and the lowest p-value arising among all gene sets was identified. This procedure was repeated in 2000 simulations, yielding a distribution for the minimum p-value expected to arise by chance alone, given the total number of gene sets evaluated for age-biased expression. We then identified the 0.05 quantile of this distribution (P*), which corresponds to the p-value that is lower than the minimal p-value that arose in 95% of the simulation trials. Those gene sets associated with p-values less than P* are denoted by yellow symbols. (TIF) Figure S5 Zeb1, AP-2 and YY1 binding site density near the TSS is associated with decreased gene expression with age in females but not males. In each figure, blue or black symbols represent the average 40-year fold-change (old/ young) among genes with at least t binding sites in the region proximal to the annotated transcription state site (2KB upstream and 200 BP downstream of the TSS). An increasing series of thresholds (t) was used to define gene sets of successively smaller sizes (horizontal axis), and for each set, the average 40-year fold- change was calculated (vertical axis). The background color (red or green) reflects the proportion of age-increased (red) to age- decreased genes (green) with respect to a given threshold (t), where an increase in the size of the red region denotes a higher proportion of age-increased genes with increasing t, and an increase in the size of the green region denotes a higher proportion of age-decreased genes with increasing t. For any given threshold t, blue symbols denote gene sets for which the average 40-year fold- change among genes with at least t binding sites is significantly different from that of genes with fewer than t binding sites (P,0.05; two-sample t-test). The dotted horizontal line represents the average 40-year fold-change (old/young) among all 18,442 human genes included in the analysis. (TIF) Figure S6 Genomic regions with increased AP-2 binding site density are associated with decreased gene expres- sion with age in female human subjects but not male subjects. These analyses were performed with respect to all human non-coding intergenic regions (repeat-masked) (parts A– D), all intronic regions (repeat-masked) (parts E–H), and conserved sequences located 2000 BP upstream of the annotated TSS for all human genes (parts I–L). In each case, sequences were scanned to identify putative AP-2 binding sites (consensus motif: GCC----G), and length-adjusted residual scores were calculated for all sequences (see Methods). Positive scores indicate a large number of AP-2 binding sites (given sequence length) and negative scores indicate a small number of AP-2 binding sets (given sequence length). Average residual scores were then compared between a foreground set of sequences (adjacent to age-increased or age- decreased genes) and a background set of sequences (i.e., all other sequences excluding those in the foreground). The central tendency of the foreground sequence residual scores is displayed for (A, E, I) sequences within or adjacent to age-increased genes (red lines) in females, (B, F, J) sequences within or adjacent to age- PLoS ONE | www.plosone.org 16 March 2012 | Volume 7 | Issue 3 | e33204