Comparison of <i>cis</i> effects and <i>trans</i> effects between liver and retina.

<p>(A) Using the same analytic pipeline as for retina, genes in the liver were classified as conserved (yellow; largely obscured), <i>cis</i> (green), <i>trans</i> (red), or <i>cis</i> and <i>trans</i> (purple). (B) <i>Cis</i>- and <i>trans</i>- regulated genes were further subcategorized as to whether the <i>cis</i> and <i>trans</i> effects acted in the same (“+” sign; pink and brown) or opposite (“−” sign; orange and blue) directions, and whether the <i>cis</i> (CAPS; orange and pink) or <i>trans</i> (CAPS; blue and brown) effect was stronger. (C) Number of genes classified as <i>cis</i> in liver and conserved in retina, <i>cis</i> in both tissues, or <i>cis</i> in retina and conserved in liver. (D) Number of genes classified as <i>trans</i> in liver and conserved in retina, <i>trans</i> in both tissues, or <i>trans</i> in retina and conserved in liver. (E) Correlation between genes classified as <i>cis</i> in both tissues. Pearson r values for F0 samples (left) and F1 samples (right) are shown. (F) Correlation between genes classified as <i>trans</i> in both tissues. Pearson r values for F0 samples (left) and F1 samples (right) are shown. Insets, magnified view.</p

Comparison of differentially expressed and <i>cis</i>-effect genes associated with photoreceptor CREs.

<p>Genes were classified as being associated with CRX ChIP-seq peaks (CBR-associated) or not. (A) Differentially expressed (DE) autosomal genes were identified using DESeq at 5% FDR. The proportions of genes with higher expression in F0 Cast/EiJ than F0 C57BL/6J at various fold changes are shown. (B) <i>Cis</i>-effect autosomal genes were identified using MMDIFF. Proportions of genes with higher expression in F1 Cast/EiJ allele than F1 C57BL/6J allele at various fold changes are shown. P-values were calculated with two-tailed Fisher's exact test. N.S. = not significant, *<0.05, **<0.01, ***<0.001, **** <0.0001.</p

Agreement between F1 biological replicates.

<p>Agreement between F1 biological replicates.</p

Analysis of variant density in photoreceptor CREs.

<p>(A) The number of Cast/EiJ (top) or Spret/EiJ (bottom) SNPs and indels relative to C57BL/6J was determined in 50 bp windows (sliding 25 bp at a time) across the 2 kb region centered on CBRs. Phylogenetic conservation for CBRs is based on PhastCons scores as found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109382#pone.0109382-Corbo2" target="_blank">[6]</a>. The highlighted area corresponds to the central 300 bp region. (B) Histogram showing frequency of variants (SNPs+indels) in the 1 kb region centered on all CBRs (black), CBRs associated with <i>cis</i>-effect genes (green), and CBRs associated with <i>trans</i>-effect genes (red). Total bar height was normalized to 1 for each category.</p

Agreement between F0 biological replicates.

<p>Agreement between F0 biological replicates.</p

Accuracy of X chromosomal read mapping in F1 samples.

<p>Accuracy of X chromosomal read mapping in F1 samples.</p

Classification of genes by mechanism of gene regulatory divergence.

<p>(A) Genes were classified as conserved (yellow; largely obscured), <i>cis</i> (green), <i>trans</i> (red), or <i>cis</i> and <i>trans</i> (purple). (B) <i>Cis</i>- and <i>trans</i>-effect genes were further subcategorized as to whether the <i>cis</i> and <i>trans</i> effects acted in the same (“+” sign; pink and brown) or opposite (“−” sign; orange and blue) directions, and whether the <i>cis</i> (CAPS; orange and pink) or <i>trans</i> (CAPS; blue and brown) effect was stronger. Insets, magnified views.</p

Study design.

<p>(A) F0 and F1 mice were generated via the depicted crosses. The schematic diagram illustrates example expression patterns for a <i>cis</i> effect, <i>trans</i> effect, and parent-of-origin effect. For a <i>cis</i> effect, in the F1 hybrids, the Cast/EiJ allelic expression relative to the C57BL/6J allelic expression recapitulates the ratio of gene expression levels in the F0 homozygotes. For a <i>trans</i> effect, the F1 hybrids express the Cast/EiJ and C57BL/6J alleles equally. For a parent-of-origin effect, there is preferential expression of the maternal allele (as depicted) or the paternal allele, as seen by comparison of the reciprocal F1 hybrids. (B) An overview of the workflow is shown.</p

<i>Cis</i>-effect genes associated with retinal disease and photoreceptor CREs.

<p>(A) <i>Cis</i>-effect genes associated with CRX ChIP-seq peaks were matched against the RetNet database of retinal disease genes. The yellow circle highlights <i>Sag</i>. (B) <i>Sag</i> locus, mm9. Top: Screenshot from UCSC Genome Browser <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109382#pone.0109382-Kent1" target="_blank">[77]</a>. DNaseI hypersensitivity site (DHS) signals are from ENCODE data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109382#pone.0109382-ENCODE1" target="_blank">[8]</a>. Bottom: Enlargement of boxed region. The 0.7 kb promoter region is depicted here. Locations of known Cast/EiJ variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109382#pone.0109382-Keane1" target="_blank">[19]</a> are depicted as green tic marks (SNPs) or blue tic marks (indels). (C) Retinal explant electroporation was used to assay the activity of the 0.7 kb <i>Sag</i> promoter region of B6 and Cast alleles. Representative images are shown here for the B6 (top) and Cast (bottom) 0.7 kb <i>Sag</i> promoter constructs driving DsRed expression. <i>Rho</i>-CBR3-eGFP served as the loading control. (D) Quantification of the <i>cis</i>-regulatory activity measured by the explant electroporation assay. Error bar represents SEM. P-value was calculated with one-tailed Wilcoxon rank-sum test.</p