Epigenetics, Signals, and Oxytocin and Vasopressin Gene Expression in the CNS.

我们正在解决两个关键问题和相关的假设有关的表观遗传和信号转导机制，调节细胞特异性OT和VP基因在下丘脑的表达。 第一个关键问题是评估SON中OT和VP基因的染色质状态。这里的假设是，这两个基因中的某些染色质修饰在OT和VP神经元表型中特异性表达，更具体地说，SON中OT和VP的细胞特异性表达是由于VP细胞中OT基因和OT细胞中VP基因上的组蛋白的乙酰化和甲基化。这种类型的染色质修饰是非常动态的，因此是可逆的，因此在某些情况下可以在SON中发生OT和VP的共表达（参见格拉斯哥等人，1999; Xi等人，1999）。为了解决这一假设，我们现在通过以下方式进行表观遗传学研究：1）在体内各种生理条件下分析SON中OT & VP染色质和其他特定基因中的组蛋白乙酰化和甲基化模式，以及2）测试乙酰化/脱乙酰化抑制剂对OT & VP染色质和hnRNA转录的影响。另一个假设是，OT和VP基因在HNS中的细胞特异性表达和这些基因在大多数脑区域如皮质、纹状体、小脑等中的沉默，以及可能在SCN中的OT的沉默，是由于这些基因在非表达细胞中的DNA甲基化（例如，如在印记中）。OT和VP基因在诸如大脑皮层等区域中的稳定沉默，以及OT在SCN中的可能的稳定沉默可能是由于DNA甲基化，其动态性低得多，并且在许多情况下是不可逆的（例如，在印迹中）。 正在考虑进行实验来检验这一假设。 第二个关键问题是确定哪些神经信号、信号转导和转录机制调节SON中OT和VP基因的表达。来自我们实验室的先前研究表明，河豚毒素（TTX）的立体定位Alzet微渗透泵递送阻断SON中免疫反应性c-fos和VP hnRNA的急性全身性高渗诱导（Shahar et al，2004，川崎et al，2009），这表明突触输入是VP基因表达的渗透调节的必要信号。急性全身性高渗刺激增加SON中的VP但不增加OT hnRNA，尽管两种肽同样增加神经叶的分泌（Yue et al，2008 a; 2008 b），进一步表明OT与VP表型中的兴奋-分泌与兴奋-转录偶联机制存在显著差异。此外，虽然直接的神经递质刺激是必要的，但它可能不足以调节基因表达。川崎et. al（2009）表明，无论是谷氨酸，通过NMDA和AMPA受体作用。已知在急性高渗刺激期间增加HNS的VP分泌中是关键的，MCN的直接渗透刺激本身也不能增加SON中的VP基因表达。鉴于这些数据，我们假设，参与SON中VP基因表达的急性渗透诱发刺激的信号转导级联涉及最终作用于CREB和相关转录因子的特异性钙和/或cAMP依赖性细胞内激酶和/或磷酸酶的激活。正在进行的相关实验是：1）评估直接应用推定的第二信使（例如，钙离子和cAMP）对VP转录的影响; 2）各种激酶抑制剂和激酶siRNA对VP转录的急性渗透刺激的影响。

We are addressing two key issues and associated hypotheses related to epigenetic and signal-transduction mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. The first key issue is to evaluate the chromatin status of the OT & VP genes in the SON. Here the hypothesis is that certain chromatin modifications in the two genes are specifically expressed in the OT and VP neuronal phenotypes .More specifically, that the cell specific expression of OT and VP in the SON is due to the acetylation and methylation of histones on the OT gene in VP cells & the VP gene in OT cells. This type of chromatin modification is very dynamic & therefore reversible, so coexpression of OT & VP in the SON could occur under some circumstances (see Glasgow et al, 1999; Xi et al, 1999.) To address this hypothesis, we are now doing epigenetic studies by:1) analysing histone acetylation & methylation patterns in OT & VP chromatin in SON and in other specific genes, under various physiological conditions in vivo, and 2) to test effects of acetylation/deacetylation inhibitors on OT & VP chromatin and hnRNA transcription in vivo. An additional hypothesis is that the cell-specific expression of the OT & VP genes in the HNS and the silencing of these genes in most brain areas such as in cortex, striatum, cerebellum, etc, and possibly also for OT in the SCN, is due to the DNA methylation of these genes in the non-expressing cells (e.g, as in imprinting). The stable silencing of the OT & VP genes in areas such as cerebral cortex, etc, and possibly of OT in the SCN could be due to DNA methylation, which is much less dynamic and in many cases irreversible (e.g, in imprinting). Experiments are under consideration to test this hypothesis. The second key question is to determine which neural signals, signal transduction, and transcriptional mechanisms regulate OT and VP gene expression in the SON. Previous studies from our laboratory showed that stereotaxic Alzet miniosmotic pump delivery of tetrodotoxin (TTX) blocks acute systemic hyperosmotic induction of immunoreactive c-fos and VP hnRNA in the SON (Shahar et al, 2004, Kawasaki et al, 2009) which suggests that synaptic input is an essential signal for osmotic regulation of VP gene expression. Acute systemic hyperosmotic stimuli increases VP but not OT hnRNA in the SON , although both peptides equally increase in secretion from the neural lobe (Yue et al, 2008a; 2008b) further suggesting a profound difference in excitation-secretion vs excitation-transcription coupling mechanisms in the OT vs VP phenotypes. In addition, while direct neurotransmitter stimulation is necessary it may not be sufficient to regulate gene expression. Recent studies by Kawasaki et. al (2009) showed that neither glutamate, acting via NMDA and AMPA receptors. known to be critical in increasing VP secretion by the HNS during acute hyperosmotic stimulation, nor direct osmotic stimulation of the MCNs are able by themselves to increase VP gene expression in the SON. Given these data we hypothesize that The signal transduction cascade involved in acute osmotic evoked stimulation of VP gene expression in the SON involves activation of specific calcium and/or cAMP-dependent intracellular kinases and/or phosphatases acting ultimately on CREB and related transcription factors. Related experiments in progress are to: 1) Evaluate effects of direct application of putative second messengers (e.g, calcium ion & cAMP) on VP transcription & 2) effects of various kinase inhibitors and kinase siRNAs on the acute osmotic stimulation of VP transcription.