Comparison of species-specific DHS to independently derived cells.

<p>Human DHS gains show a high level of overlap to DHS regions identified in (a) three independently analyzed human fibroblast cell lines and (b) 5 independently analyzed human LCL samples, compared to human DHS losses. Common DHS are also similarly detected.</p

Functional mutations associated with DHS gains and losses.

<p>(a–e) Scatterplots showing the enrichment of AP1 motif matches in species with increased hypersensitivity. Each “x” represents a single DHS site. (a–c) positive values on each axis indicate better motif matches on the human branch. For these regions, points in the upper-right quadrant are regions where the AP1 motif scores better in human than either chimp or macaque, where the lower left represent AP1 motif scores worse in human. The number of DHS sites in these quadrants are indicated. (d–e) For chimp gain and loss regions, positive values for each axis indicate a better motif match in the chimp branch. (f) The AP1 motif from JASPAR and an example alignment of a representative human gain region representing a point along the diagonal in the upper-right quadrant in panel a. (g) Boxplots summarizing the results from AP1 and three other motifs. The boxplots show the distribution of (combined) log-ratios (relative to the appropriate species). <i>P</i> values for differences relative to common regions are significant (asterisk) in all 4 comparisons: human DHS gains, <i>P</i><10⁻³¹; human DHS losses <i>P</i><10⁻³; chimp DHS gains, <i>P</i><10⁻¹³; chimp DHS losses, <i>P</i><10⁻⁸ (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002789#s4" target="_blank">Materials and Methods</a>). In AP1, the significant trends illustrate the same principal observed in panels a–e. Most other transcription factors have plots that show no pattern in motif score among species, such as SP1 and SOX10 (Supplemental data file 3 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002789#pgen.1002789.s001" target="_blank">Dataset S1</a>). ZEB1, a transcriptional repressor, displays an inverse relationship with hypersensitivity.</p

Comparison of DHS sites and DGE-seq data across species.

<p>(a) Analysis pipeline. DNase-sequences from each species were aligned to the native genome and lifted over to the human genome for analysis. Regions are filtered at various steps of the analysis to remove alignment and orthology artifacts (Materials and Materials). Correlation plots of DNase-seq signals (b) and DGE-seq signals (c) expression data show that both chromatin and expression data from human (Hu), chimpanzee (Ch), and macaque (Ma) are more highly correlated between biological replicates from the same tissue within a single species. Additionally, the same cell type from different species is more similar than different cell types from the same species.</p

Identification of species-specific differences in DHS sites.

<p>Species-specific DHS sites were identified by edgeR (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002789#s4" target="_blank">Materials and Methods</a>). Boxplots show the distribution of number of reads per sample in 300 bp windows. For human DHS gains (a), the 3 human samples are all significantly more open than the other 2 species. Likewise, human DHS losses (b) show lower signal in human compared to both chimpanzee and macaque. A representative sampling of distributions from all DHS is shown in (c), as well as Common DHS sites (d) found in all three species that are matched for signal intensity compared to human DHS gains and human DHS losses. (e) Distribution of species-specific DHS Gains and DHS Losses relative to promoters, introns, 3′ UTR, and intergenic regions. (f) Representative screen shots of human-specific DHS Gains and Losses compared to a Common region.</p

Comparison of human DHS site gains and losses to DNase-seq data from other human cell types.

<p>The log of the DNase-seq signal intensity value, defined as the maximum parzen score (output of F-seq) for each of the coordinates that are represented along the x-axis, are represented as a heatmap in these figures. The color red represents a higher score, and thus a relatively higher DNase-seq signal, and the color blue represents a lower score. (a) 836 DHS sites were identified as differentially open (human DHS gain) in human fibroblasts compared to chimpanzee/macaque fibroblasts. These regions from Human Fibroblasts (Hu Fibro 1–3) were compared to DNase-seq data generated from 27 other human cell types (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002789#pgen.1002789.s019" target="_blank">Table S3</a>). Additional human skin fibroblast samples (listed in black) are highly similar, while some non-fibroblast cell types show less but substantial overlap and the remaining cell types show much less overlap. Only a small fraction of DHS sites were active in all 27 cell lines (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002789#pgen.1002789.s006" target="_blank">Figure S5</a>). Sites with evidence for positive selection are indicated in the horizontal bar above the heatmap. The distribution appears roughly uniform. (b) 286 DHS sites identified as differentially closed (human DHS loss) compared to chimp and macaque fibroblasts. (c) DNase-seq signal values for Common regions representing DHS sites in all three species. More than 50% of Common regions are also DHS sites in other human tissues. (d, e, f) DNase-seq values for same regions as (a, b, c), but DNase data is from orthologous region from chimpanzee and macaque fibroblasts.</p