We therefore predicted that the reduced cytosine methylation in the adult offspring of high-LG compared with low-LG mothers would result in greater NGFIA binding to the exon 17 promoter. This prediction was confirmed using a ChIP assay (described above) examining in vivo formation of protein-DNA complexes in hippocampal tissue from adult animals.67 The results indicated a threefold greater binding

Inhibitors,research,lifescience,medical of NGFIA protein to the hippocampal exon 17 GR promoter in the adult offspring of high-LG compared with low-LG mothers. Using the same tissue samples and an antibody against the acetylated form of H3, we67 found dramatically increased acetylated H3 association with the exon 17 GR promoter in the offspring of the high-LG mothers. As described above, histone acetylation is CYT387 clinical trial associated with active states of gene expression. Inhibitors,research,lifescience,medical These findings are therefore consistent with the idea of increased NGFIA binding to the exon 17 promoter, enhanced histone acetylation, and increased GR transcriptional activation. We confirmed that DNA methylation inhibits the ability of NGFIA to activate Inhibitors,research,lifescience,medical the exon 17 promoter using a transient cotransfection assay in HEK293

cells. The HEK293 cells are not of neural origin and thus allow us to measure the transcriptional consequences of interaction of NGFIA with either a methylated or nonmethylated version of the GR exon 17 promoter per se, independent of the complications associated with other neuronal signals. We used transfection technology to introduce into the HEK cells (i) a viral vector containing the NGFIA gene, Inhibitors,research,lifescience,medical to produce a intracellular signal usually inactive in HEK cells; and (ii) an exon 17-luciferase reporter construct. This genomic construct that included the exon 17 promoter sequence

fused with a luciferase reporter gene (the level of Inhibitors,research,lifescience,medical the easily measured luciferase activity is used as a measure of exon 17 promoter activity). Cotransfection of the NGFIA expression vector significantly increases luciferase activity; however, this effect is dramatically reduced if the CpG dinucleotides within the exon 17 sequence are methylated. Moreover, the effect of NGFIA on transcription through an exon 17-luciferase reporter construct was almost completely abolished with a point mutation at the 5′ cytosine (a cytosine to adenosine mutation). Taken together, these findings suggest that an “epimutation” at a single cytosine within the NGFIA consensus no sequence alters the binding of NGFIA and might therefore explain the sustained effect of maternal care on hippocampal GR expression and HPA responses to stress. How does maternal care alter cytosine methylation? Maternal behavior could either inhibit de novo methylation or stimulate demethylation. To address this question, we67 performed a simple developmental study of the methylation pattern of GR exon 17 promoter from embryonic day 20 to day 90 (a fully, sexually mature adult rat).