Epigenetics is defined as the covalent modification of chromatin that influences activity-dependent changes in gene expression. The two main molecular epigenetic mechanisms are posttranslational histone modifications and DNA methylation. Our working hypothesis is that chromatin modification by DNA methylation of the OT and VP genes and their respective receptor genes is responsible for the selective expression of these genes in the brain. Methylation of DNA is a direct chemical modification of a cytosine catalyzed by a class of enzymes known as DNA methyltransferases (DNMTs). The DNMTs transfer methyl groups to cytosine residues, specifically at the 5th position of the pyrimidine ring. Cytosines must be immediately followed by a guanine to be methylated. These CpG dinucleotide sequences are highly underrepresented in the genome, and often occur in small clusters known as CpG islands. Of the three main DNMTs - 1, 3a and 3b, the latter two are thought to be responsible for de novo methylation on previously unmethylated CpG sites. Hypermethylation of CpG islands in the vicinity of genes is usually considered to be a transcription suppressing mechanism, although it was shown in some cases to be associated with transcription activation. The transcription regulating role of DNA methylation is mediated by methyl-DNA binding proteins (MBDs) such as MeCP2 whose loss of function is responsible for Rett syndrome. Of all the epigenetic mechanisms, DNA methylation is considered as the most stable, thus most suitable for long-term processes underlying maintenance and persistence of memory. Indeed, inhibiting brain DNMTs activity alters DNA methylation, blocks hippocampal LTP and impairs hippocampal-dependent memory formation. Recent studies have shown that DNA methylation is more dynamic than previously thought due to active demethylation by enzymes such as Gadd45b. DNA methylation was suggested to be involved in the regulation of the oxytocinergic system in the brain since the OT receptor gene was found to be hypermethylated on its promoter-located CpG island in the prefrontal cortex of autistic individuals. Since we proposed that there was a potential regulation of OT and VP expression by DNA methylation, we studied the differential methylation of specific CpG islands within the OT and VP genes in different brain areas to test this hypothesis. The results indicated that there were no differences in methylation patterns between the OT and VP genes in the CpG islands that we studied, when comparing DNA from brain regions that do not express these peptide genes (e.i., cortex) versus brain regions that do (i.e, in the SON). However, our studies on the oxytocin receptor (OXTR) gene did implicate methylation in the regulation of its gene expression. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor (OXTR), which is the product of a single gene. Transcription of the OXTR is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. Here, we hypothesized that methylation of the promoter of the OXTR gene (OXTRp) modulates its transcription, in a manner that is subject to physiological changes. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between OXTR transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of OXTR transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the OXTRp was found to be differently correlated with OXTR expression in mammary glands and uterus of virgin and post-partum mice, suggesting that it plays a distinct role in OXTR transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that epigenetic regulation by DNA methylation of the OXTR gene promoter does modifiy expression of the OXTR gene. This project will terminate in the next fiscal year.
| Gainer, H (2012) Cell-type specific expression of oxytocin and vasopressin genes: an experimental odyssey. J Neuroendocrinol 24:528-38 |
| House, Shirley B; Li, Congyu; Yue, Chunmei et al. (2009) Effects of ciliary neurotrophic factor and leukemia inhibiting factor on oxytocin and vasopressin magnocellular neuron survival in rat and mouse hypothalamic organotypic cultures. J Neurosci Methods 178:128-33 |
| Kawasaki, Makoto; Ponzio, Todd A; Yue, Chunmei et al. (2009) Neurotransmitter regulation of c-fos and vasopressin gene expression in the rat supraoptic nucleus. Exp Neurol 219:212-22 |
| Scordalakes, Elka M; Yue, Chunmei; Gainer, Harold (2008) Experimental approaches for the study of oxytocin and vasopressin gene expression in the central nervous system. Prog Brain Res 170:43-51 |
