within the shared set of genes that were down-regulated in children with autism and children of older fathers (figure 4c). Importantly, we also found that the same pathways were enriched in the lists of genes that were significantly down-regulated but not shared between children with autism and children of older fathers (Table S2), i.e. there was overlap in biological pathways even when the specific genes were not identical. Significant pathways associated with up-regulated genes (Table S3) were fewer in number and did not appear to offer any mechanistic understand- ing for global changes in gene expression regulation. Pathway analysis of gene lists obtained using more stringent cutoffs (51% present and a 1.25-fold change) highlighted many of the same pathways related to transcriptional regulation even though the gene lists were much shorter (72 compared to 593 genes in the list of overlapping down-regulated genes) (table S4). Cross-validation (figure S1 and table S1) To evaluate the robustness of our findings, experimental groups were sub-divided to perform statistical cross-validation. For instance, the group of 82 children with autism was divided into 2 groups of 41 children that were balanced for paternal age and microarray scan batch (Autism1 and Autism2), the group of children with older fathers (.30 years old) was divided into 2 groups of 14 children (Old father1 and Old father2), and the group of children of younger fathers (, or = 30 years old) was divided into 2 groups of 14 children (Young father1 and Young father2). With respect to variance in gene expression (Figure S1), cross- validation of autism vs. children of young fathers remained significant in both analyses, whereas the difference between children of older fathers and children of younger father was no longer significant (Figure S1). Cross-validation using Pearson correlations found that paternal age was significantly correlated within the Conrol1 group (p = .02) but not in the Control2 group. The lack of significance in the cross-validation of paternal age effects appeared to be related to decreased power because of the smaller number of subjects per group in the analysis of age effects, as the means of the subgroups were similar to those of children with autism (figure S1). Cross-validation of pathway analysis indicated that these findings were highly robust. Significant gene lists from subgroups (Autism1 vs. Young father1, Autism2 vs. Young father2, Old father1 vs. Young father1, and Old father2 vs. Young father2) indicated that in all cases, significantly down-regulated genes in children with autism or children of older fathers were enriched for genes involved in transcription (Table S1). It was also noted in these analyses that, in all cases, sets of down-regulated genes were also enriched for zinc finger C2H2 proteins and for genes involved in zinc pathways. Zinc has a well-documented role in neurode- velopment[36,37,38] and also plays any important role in the stabilization of many proteins involved in transcription[38], suggesting that zinc-mediated effects on transcription may be particularly important in contributing to global effects on gene expression. Inhibition of transcription leads to a decreased variance in gene expression (figure 5) Pathway analysis showed that regulators of transcription were enriched in the subset of genes that were down-regulated in children with autism and control children of older fathers. This suggested that dysregulation of transcription might be a mecha- nism underlying the decreased variance in gene expression in these groups of children. To explore this hypothesis, we used a publically available dataset to examine whether inhibition of transcription can lead to decreased variance in gene expression. In the gene Autism Changes Global Gene Expression Regulation Figure 5. Transcription inhibitors lead to decreased variance in gene expression. Gene expression data from a publically available dataset show that treatment of a lung cancer cell line with actinomycin D and a high dose of sapphyrin results in decreased variance in gene expression compared to treatment with mannitol or a low dose of sapphyrin. Error bars represent standard error. doi:10.1371/journal.pone.0016715.g005 expression study, a human lung cancer cell line, A549, was treated with transcription inhibitors for four hours. Treatment with high levels of sapphyrin (n = 3, p = .07), a new class of transcription inhibitor, or actinomycin D (n = 3, p = .04) led to a decrease in variance in gene expression when compared to control treatment with mannitol (figure 5). Low levels of sapphyrin had no effect on variance in gene expression. When study groups were pooled to increase power, actinomycin D (n = 3) + high sapphyrin (n = 3) vs. low sapphyrin (n = 3) + mannitol, (n = 3), the effect of transcription inhibition was a highly significant decrease in variance in gene expression (p = .003) (figure 5). Discussion Studying the variance in the distribution of gene expression represents a new approach to gene expression analysis. Because changes in a limited number of genes could not account for a change in the shape of the entire gene expression distribution, changes in variance suggest alterations at global levels of gene expression regulation. This ‘‘birds-eye view’’ of gene expression levels contrasts with gene-centered approaches that seek to identify specific genes or pathways that differ between groups. While gene specific approaches are useful they may miss changes occurring at the global level of gene expression regulation. The two approaches, therefore, represent distinct yet potentially compli- mentary approaches to understanding the full magnitude of effects on gene expression regulation. Here we used our novel approach PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e16715