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Figure 4. Retention and functional differentiationa. (left) Comparison of L and S gene loss by KEGG categories. X-axis is fraction loss of L genes, and the y-axis is fraction loss of S genes. Blue line is expected L/S loss based on genome-wide average (56.4%). Red points are functional categories that show a high degree of loss (χ2 test p<0.01). Magenta points are functional categories that show a high degree of retention (p<0.01). (right) Similar scatterplot for tissue WGCNA categories. See Supplemental Note 10.1 for a more detailed discussion.b. Boxplot of log10(Ltpm/Stpm) for homeologous gene pairs, zoomed in to show medians. Ovary and maternally-controlled developmental time points are on the left (light blue and dark blue bars respectively), zygotically-controlled developmental time points and adult tissues are on the right (red and green bars respectively). The red line shows the equal ratio log10(1). On average maternal data sets express the L gene of a homeologous pair 12% higher than as S (median = 0%), while the zygotic tissues and time points express the L gene of a homeologous pair 25% higher than S (median = 1.8%). The difference between the mean and medians is explained by many genes with large differences between homeologs, illustrated by the full distribution in Extended Data Fig. 8c. Here, to illustrate the difference in median of zygotic expression, we zoom in on the center of the boxplot.c. (left) Developmental expression plot and (right) epigenetic landscape surrounding hoxb4. L expression is red, S expression is blue, tropicalis expression is shown in black. The right panel shows the genomic profiles of H3K4me3 (green) and p300 (yellow) ChIP-seq tracks, as well as DNA methylation levels determined by whole-genome bisulfite sequencing (grey). The gene annotation track shows the hoxb4 gene on L (top) and S. The conservation between the L and S genomic sequence is shown in grey between the gene annotation tracks.d. (left) Developmental expression plot and (right) epigenetic landscape surrounding numbl. L expression is red, S expression is blue, tropicalis expression is shown in black. The small amount of expression seen in maternal numbl and numbl.L is consistent between replicates. In addition to the tracks described for c), the right panel shows RNA Polymerase II (RNAPII; purple) and H3K36me3 (blue) ChIP-seq profiles.e. Representative embryos with GFP expression driven by either six6.L-CNE or six6.S-CNE linked to a basal promoter-GFP cassette (six6.L-CNE:GFP and six6.S-CNE:GFP, respectively). GFP expression was detected by in situ hybridization. Semi-quantitative image analysis revealed a statistically significant difference in their average expression level (p < 0.01); the expression driven by six6.S-CNE (n = 27) was 0.6-fold weaker than that by six6.L-CNE in the eye region (n = 32). Given eye-specific patterns of their endogenous expression, the six6 genes likely have additional silencers for restricting enhancer activity of the CNEs in the eye.
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