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，它影响基因表达的活性依赖变化。两种主要的分子表观遗传机制是组蛋白翻译后修饰和DNA甲基化。因此，本项目的关键问题是评估SON中OT和VP基因的染色质状态。 我们的工作假设是，这两个基因中的某些染色质修饰在OT和VP神经元表型中特异表达。我们先前试图研究的一个特殊的表观遗传学机制是组蛋白在OT-和VP-MCNs的OT和VP基因上的乙酰化和甲基化。这种类型的染色质修饰是非常动态的，因此是可逆的，因此在某些情况下，在SON的一些MCNs中观察到OT和VP的条件性共表达。为了解决这一假设，我们进行了各种表观遗传学实验，重点分析了不同生理条件下SON中OT和VP染色质以及其他特定基因的组蛋白乙酰化和甲基化模式，并在体内测试了乙酰化/去乙酰化抑制剂对OT和VP染色质和hnRNA转录的影响。不幸的是，这些实验没有成功，很大程度上是因为传统的组织打孔技术为了分离染色质而采集SON组织产生了太多来自非MCN细胞的污染染色质，从而为后续的芯片方法造成了不利的信噪比情况。类似的实验正计划在来年进行，将使用来自SON的激光显微解剖(LCM)MCN，以努力改善芯片协议的信噪比情况。 我们正在研究的第二个特定的表观遗传机制是DNA甲基化。DNA甲基化是一类被称为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。胞嘧啶之后必须紧跟要甲基化的鸟嘌呤。这些CpG二核苷酸序列在基因组中的代表性很低，通常出现在称为CpG岛的小簇中。在三个主要的DNMT-1、3a和3b中，后两个被认为是负责先前未甲基化的CpG位点上的从头甲基化。基因附近CpG岛的高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与转录激活有关。DNA甲基化的转录调控作用是由甲基DNA结合蛋白(MBDS)介导的，如MeCP2，其功能丧失与Rett综合征有关。 在所有的表观遗传机制中，DNA甲基化被认为是最稳定的，因此最适合于记忆维持和持久性的长期过程。事实上，抑制大脑DNMTS活性会改变DNA甲基化，阻断海马体LTP，并损害海马体依赖记忆的形成。最近的研究表明，由于Gadd45b等酶的活性去甲基化，DNA甲基化比以前认为的更有活力。DNA甲基化被认为参与了脑内催产素能系统的调节，因为在自闭症患者的前额叶皮质中，发现OT受体基因在其启动子位置的CpG岛上发生了高甲基化。 我们已经筛选了大鼠OT/AP基因组区域的CpG岛。有趣的是，我们发现这两个基因在它们的序列中都包含一个CpG岛，但位于不同的位置。OT CpG岛包括所有三个外显子和一些5‘非翻译区，而VP CpG岛只包括外显子1-2和一些5’非翻译区。这些基因中都存在一个CpG岛，这表明DNA甲基化可能参与了它们的转录调控，而CpG岛相对于编码序列的不同位置表明，DNA甲基化可能对OT和VP的表达进行了差异调控。 我们假设，OT/VP基因组区域内CpG岛的差异甲基化参与了这些基因表达模式的控制。在目前正在进行的验证这一假设的实验中，我们使用我们开发的AAV-LCM策略的立体定位注射，并在项目编号1 Z01 NS002723-25 LNC的摘要中描述，以便识别和分离OT-和VP-MCNs用于RNA分析。然而，在这种情况下，我们从已鉴定的OT-和VP-MCNs池中分离DNA，用于通过硫酸氢盐转换程序分析它们的甲基化模式。这些实验需要更多数量的LCM分离的MCNs，而不是那些直接用于RNA定量聚合酶链式反应的MCNs，我们目前正在收集这一目的所需的神经元数量。 本实验室在下丘脑-神经垂体系统(HNS)和SCN神经元中建立了小鼠和大鼠大细胞神经元(MCNs)的器官型培养，为研究OT和VPMCN表型的各种分子和生理模型提供了有价值的实验模型。我们一直在使用这些培养物来评估不同的DNMT抑制剂(如ZeBularine或5-aza-deoxcytidine)对SON中OT和VP基因的甲基化和转录的影响。这些结果将确定SON中甲基化过程的动态程度，并将直接评估其对下丘脑OT/VP转录的影响。

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. Therefore, the key issue in this project is to evaluate the chromatin status of the OT and VP genes in the SON. Our working hypothesis is that certain chromatin modifications in the two genes are specifically expressed in the OT and VP neuronal phenotypes. One specific epigenetic mechanism that we previously attempted to study is the acetylation and methylation of histones on the OT and VP genes in the OT- and VP-MCNs. This type of chromatin modification is very dynamic & therefore reversible, so the conditional co-expression of OT & VP observed in some MCNs in the SON could occur under some circumstances. To address this hypothesis, we did various epigenetic experiments focused on analyzing specific histone acetylation & methylation patterns in OT & VP chromatin in SON and in other specific genes, under various physiological conditions in vivo, and tested effects of acetylation/deacetylation inhibitors on OT & VP chromatin and hnRNA transcription in vivo. Unfortunately, these experiments were not successful, in large part, because the harvesting of SON tissues for the isolation of chromatin by conventional tissue punch techniques produced too much contaminating chromatin from non-MCN cells thereby creating an unfavorable signal to noise situation for the subsequent ChIP methodology. Similar experiments are being planned for the coming year, which will use laser microdissected (LCM) MCNs from the SON in an effort to improve the signal to noise situation for the ChIP protocol. The second specific epigenetic mechanism that we are studying is DNA methylation. 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. We have screened the rat OT/AP genomic area for CpG islands. Interestingly, we found that both genes include a CpG island in their sequence, but in distinct locations. While the OT CpG island encompass all three exons plus some of the 5' untranslated area, the VP CpG island include only exons 1-2 with some of the 5' untranslated area. The existence of a CpG island within each of these genes suggests the possible involvement of DNA methylation in their transcriptional regulation, and the distinct locations of the CpG islands relative to the coding sequence suggests a potential differential regulation of OT and VP expression by DNA methylation. We hypothesize that differential methylation of CpG islands within the OT/VP genomic area is involved in the control of the expression pattern of these genes. In experiments presently under way to test this hypothesis we use the stereotaxic injection of AAV-LCM strategy that we developed and describe in the summary of project No. 1 Z01 NS002723-25 LNC in order to identify and isolate OT- and VP-MCNs for RNA analysis. However, in this case we are isolating DNA from the pools of the individual identified OT- and VP-MCNs for analysis of their methylation patterns by the bisulfate conversion procedure. These experiments require much larger numbers of LCM-isolated MCNs than those directed at RNA analysis by qPCR, and we are presently collecting the required number of neurons needed for this purpose. Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) and SCN neurons have been developed in our lab and serve as valuable experimental models for various molecular and physiological studies of the OT and VP MCN phenotypes. We have been using these cultures to evaluate the effects of various DNMT inhibitors (e.g, zebularine or 5-aza-deoxycytidine) on methylation and transcription of the OT, and VP genes in the SON. These results will determine how dynamic the methylation process in the SON and will directly evaluate its effect on OT/VP transcription in the hypothalamus.