Figure 7.

Location of key methylation sites in TDO2 (marked with red asterisks): within alternative exon 1 (Alt-1), in two of the three glucocorticoid motifs and just upstream of exon 1 in the promoter region. Methylation of the GRE sites should enhance binding of the glucocorticoid receptor protein, docking of cortisol to the site [195], and act to increase expression of the gene. The CEBP-β transcription factor motif identified by Kudo et al. [198] enhances GRE motif effects when both are bound by their respective ligands [197]. Hypomethylation within Alt-exon 1 (cg15736994; see iMethyl for location) is observed in children who are economically underprivileged [189], and would likely diminish inclusion of the alternative transcript from this location, as methylation within alternative exons can act to enhance inclusion in the final transcript [77]. However, the absolute level of expression may be higher when the hypomethylation is in the first exon [16], thus the net effect cannot be estimated. Hypermethylation of a CpG site just upstream of exon 1 was reported for newborns of mothers who smoked during pregnancy [204]. Chronic mild stress in adult rodents has been shown to decrease methylation in the general region of the promoter [205]. Hypermethylation at a CpG site in intron 3 is found in placental tissue of pregnant women who worked on the nightshift [200]. The transcription factor NF-kB is predicted by the program Alibaba2 to bind at this site, and among other roles, it is involved in response to immune stimuli as well as stress from sleep deprivation [201,206], certainly consistent with nightshift work. Methylation of this NF-kB site would be expected to block NF-kB binding to the motif [202]. Binding of NF-kB to introns has been shown for other genes, e.g., ICAM-1, where its role is generally to stimulate expression [203].