Fig. 5: DNA methylation and expression pattern of NFIX in modern and archaic humans.

a Methylation levels along NFIX, color-coded from green (unmethylated) to red (methylated). Methylation levels around the two AMH-derived DMRs (#24 and #167) are shown in the zoomed-in panels. These two DMRs represent the regions where the most significant methylation changes are observed, but hypermethylation of NFIX in AMHs can be seen throughout the entire gene body. Chimpanzee and present-day samples were smoothed using the same sliding window as in ancient samples to allow easier comparison. The inferred schematic regulatory evolution of NFIX is shown using a phylogenetic tree to the left of the top panel. Star marks the shift in methylation from unmethylated (green) to methylated (red). b Methylation levels in DMRs #167 and #24 vs. expression levels of NFIX across 22 AMH tissues (gray). In both DMRs, higher methylation is significantly associated with lower expression of NFIX. Ust’-Ishim, Bone1 and Bone2 methylation levels (red) are plotted against mean NFIX expression across 13 osteoblast lines. Neanderthal and Denisovan methylation levels (green) are plotted against their predicted expression levels, based on the extrapolated regression line (dashed). Standard errors are marked with dotted lines. The expression levels of NFIX in Neanderthal and Denisovan shown in these graphs are extrapolated (green dots). c Box plots of expression levels of NFIX, COL2A1, ACAN, SOX9, and XYLT1 in 89 AMH samples (red) and four mouse samples (green) from appendicular bones (limbs and pelvis). Central line shows mean, box borders show 25th and 75th percentiles, whiskers extend to the most extreme data points not considered outliers, crosses show outliers. Expression levels were converted to percentiles based on the level of gene expression compared to the rest of the genome in each sample. t-test P-values are shown for each gene.