Such hypermethylation provides a likely mechanism for the suppression of Npas4 the Tet1KO mouse brain, as well as for the downregulation of Npas4 target genes, including c-Fos. We also found that both mRNA
HA-1077 in vitro and protein levels of Npas4 and c-Fos were downregulated in the cortex and hippocampus of Tet1KO mice compared to controls following memory extinction training. Postextinction methylation analysis of Npas4 promoter region indicated that its methylation remained low in the cortex and hippocampus in wild-type mice but stayed at much higher levels in Tet1KO animals, strongly correlating with Npas4 expression. We should note that locus-specific methylation and hydroxymethylation analysis in this study was performed using promoter areas of specific genes. However, we realize that methylation and hydroxymethylation of gene body as well as various distant regulatory elements may also contribute
to gene expression ( Bergman and Cedar, 2013). Future studies aimed at genome-wide methylation and hydroxymethylation analysis of the brains of Tet-deficient mice may provide a more comprehensive coverage of multiple methylation domains regulating expressions of specific find more genes. One plausible explanation for extinction impairment observed in Tet1KO mice would be that the memory extinction paradigm does not provide a sufficiently strong stimulus to overcome Tet1 deficiency. However, a stronger stimulation, such as Pavlovian fear conditioning employed during memory acquisition, may recruit additional transcriptional machinery, including other Tet proteins, thus compensating for the lack of Tet1 (Figure S5). In favor of such hypothesis is the result of our direct comparison of Npas4 and c-Fos expression in control and Tet1KO mice under various experimental conditions ( Figure 5E). Further studies using double and triple Tet knockouts should help us better understand the relative contribution and potential co-operation of different Tet proteins in various aspects of activity-driven chromatin
regulation, cognitive behavior, and plasticity. It has recently been shown that Tet1 promotes 5mC hydroxylation and active DNA demethylation Thiamine-diphosphate kinase in the adult mouse brain. Tet1 is both necessary and sufficient for neuronal activity-induced DNA demethylation of the Bdnf IX and Fgf1B promoters in the dentate gyrus ( Guo et al., 2011a and Guo et al., 2011b). It remains to be seen whether such region- and gene-specific activity-dependent DNA demethylation occurs in a more general fashion in the mammalian brain, and whether Tet1 activity is critical. Our findings do not contradict the hypothesis of a key role of Tet1 in activity-induced DNA demethylation, but they also suggest a maintenance-type role for neuronal Tet1, perhaps protecting locus-specific DNA regions from excessive methylation ( Williams et al., 2012).