Epigenetic regulation of Oxytocin and Vasopressin Gene Expression in the CNS.

表观遗传学被定义为染色质的共价修饰，影响基因表达的活性依赖性变化。两个主要的分子表观遗传机制是翻译后组蛋白修饰和DNA甲基化。我们的工作假设是，通过OT和VP基因及其各自的受体基因的DNA甲基化进行的染色质修饰负责这些基因在大脑中的选择性表达。 DNA的甲基化是由一类称为DNA甲基转移酶（DNMT）的酶催化的胞嘧啶的直接化学修饰。DNMT将甲基转移到胞嘧啶残基，特别是在嘧啶环的第5位。胞嘧啶后面必须紧跟着要甲基化的鸟嘌呤。这些CpG二核苷酸序列在基因组中的代表性非常低，并且通常以称为CpG岛的小簇出现。在三种主要的DNMT- 1、3a和3b中，后两种被认为负责先前未甲基化的CpG位点上的从头甲基化。基因附近CpG岛的超甲基化通常被认为是一种转录抑制机制，尽管在某些情况下它被证明与转录激活有关。DNA甲基化的转录调节作用是由甲基DNA结合蛋白（MBD）介导的，如MeCP 2，其功能丧失是Rett综合征的原因。 在所有的表观遗传机制中，DNA甲基化被认为是最稳定的，因此最适合于维持和持久记忆的长期过程。事实上，抑制大脑DNMT活性会改变DNA甲基化，阻断海马LTP并损害海马依赖性记忆的形成。最近的研究表明，由于Gadd 45 b等酶的活性去甲基化，DNA甲基化比以前认为的更具动态性。DNA甲基化被认为参与了大脑中催产素能系统的调节，因为在自闭症个体的前额叶皮层中发现OT受体基因在其启动子定位的CpG岛上高度甲基化。 由于我们提出OT和VP表达可能受DNA甲基化的调节，因此我们研究了不同脑区OT和VP基因内特定CpG岛的差异甲基化以验证这一假设。结果表明，当比较来自不表达这些肽基因的脑区域的DNA时，我们研究的CpG岛中OT和VP基因之间的甲基化模式没有差异（即，皮质）与大脑区域（即，在SON中）。 然而，我们对催产素受体（OXTR）基因的研究确实暗示了甲基化在其基因表达调控中的作用。 催产素的所有中枢和外周作用都是通过催产素受体（OXTR）介导的，该受体是单个基因的产物。OXTR的转录受性腺类固醇激素的调节，并且在分娩期间在子宫和乳腺中显著升高。在这里，我们假设OXTR基因（OXTRp）的启动子甲基化调节其转录，以一种方式，是受生理变化。下丘脑衍生的GT 1 -7和乳腺衍生的4 T1小鼠细胞系显示OXTR转录与基因启动子甲基化之间的负相关性，去甲基化导致4 T1细胞中OXTR转录的显著增强。使用报告基因分析，我们表明，甲基化的基因启动子中的特定位点，包括雌激素反应元件，显着抑制转录。此外，发现OXTRp的甲基化与处女和产后小鼠的乳腺和子宫中的OXTR表达存在不同的相关性，这表明它在组织之间和不同生理条件下在OXTR转录中起着不同的作用。总之，这些结果支持通过OXTR基因启动子的DNA甲基化的表观遗传调节确实修饰OXTR基因的表达的假设。 该项目将在下一个财政年度结束。

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.