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.

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