Phase I enzymes as part of an epigenetic switch

描述（由申请人提供）：该提议基于以下假设：1期酶参与胚胎开关，并且在受精时开关的默认位置是“关闭”。这将发生在一个亚组的基因，视黄酸反应元件（RARE）在其调控DNA。“OFF”位置可能是由于视黄酸受体的功能尚未得到足够的关注：在缺乏视黄酸的情况下，辅阻遏物分子与RAR？在RAREs上，correpresor阻止了RAR？与辅激活因子相互作用，并引入组蛋白去乙酰化酶，使染色质浓缩。 随着发育的进行，视黄酸通过乙醛脱氢酶被精确地解析出来，并且当它离开其细胞来源时，视黄酸可以进入相邻细胞，但受到表达特异性细胞色素p450活性（cyp 26 a1、cy 26 b1、cyp 26 c1）的细胞的边界的限制，这些细胞色素p450活性将视黄酸氧化成非功能性配体。所有这些成分都在发育中的胚胎中受到仔细调节，并具有直接和/或间接的反馈回路，这些反馈回路受到视黄酸的影响。视黄酸的分配然后解除基因的抑制并影响胚胎细胞的分化。 这意味着什么，为什么它很重要？在成人中被认为是功能性I期酶的基因在胚胎药物的开发中起着关键作用;影响这些基因或设计与它们相互作用的激素可能会在不知不觉中在胚胎发育中发挥戏剧性或微妙的作用，特别是神经系统。我们将用反义吗啉代、指示转基因胚胎和其他试剂和方法的工具箱来机械地测试这一假设，以识别受抑制的基因，并观察这些成分的实验操作是否会对种系产生跨代影响。 具体来说，我们将使用转基因视黄酸指示剂斑马鱼胚胎（在本实验室开发），以帮助我们确定哪些基因可能通过这种机制在合子基因表达启动时被抑制。我们将通过使用抗Smrt抗血清免疫沉淀斑马鱼smrt辅阻遏物相关染色质来证实这种关联。我们将比较通过这种双重筛选的基因家族与其他人发现的胚胎干细胞分化时打开的基因，我们将询问我们的400多个Agilent微阵列斑马鱼发育数据库，以确定一般发育表达模式和基因，这些基因似乎是及时共调节的。通过这项工作，我们希望不仅能验证这一假设，而且开始确定一个重要的基因子集，这些基因被这种RAR-辅阻遏物机制“抑制”

DESCRIPTION (provided by applicant): This proposal is based upon the hypothesis that phase 1 enzymes are involved in an embryonic switch and that the default position for the switch at the time of fertilization is "OFF". This would occur in a subset of genes that have retinoic acid response elements (RAREs) in their regulatory DNA. The "OFF" position would be due to a function of retinoic acid receptors that has not received sufficient notice: in the absenc of retinoic acid, corepressor molecules bind to the RAR? on the RAREs, the corepresor prevents the RAR? from interacting with a coactivator and also brings in a histone deacetylase that condenses the chromatin. As development proceeds, retinoic acid is precisely parsed out through aldehyde dehydrogenases, and as it leaves its cellular source the retinoic acid can enter adjacent cells but is limited by boundaries of cells expressing specific cytochrome p450 activity (cyp26a1, cy26b1, cyp26c1) that oxidize retinoic acid to a nonfunctional ligand. All of these components are carefully regulated in the developing embryo and have direct and/or indirect feedback loops that are affected by retinoic acid. The allotment of retinoic acid then derepresses genes and affects the differentiation of the embryonic cells. So what does this mean and why is it important? Genes recognized as functional Phase I enzymes in the adult play a critical in the development of the embryo-drugs; hormones that affect these genes or designed to interact with them could then unknowingly play dramatic or subtle roles in the development of the embryo in general and specifically, the nervous system. We will be testing this hypothesis mechanistically with a toolbox of antisense morpholinos, indicator transgenic embryos, and additional reagents and approaches to identify the genes repressed and to see if experimental manipulation of these components can have transgenerational effects upon the germline. Specifically we will be using transgenic retinoic acid indicator zebrafish embryos (developed in this laboratory) to help us identify what genes might be repressed via this mechanism at the time of zygotic gene expression initiation. We will confirm this association by immunoprecipitating zebrafish smrt corepressor associated chromatin using anti-Smrt antisera. We will compare the identified family of genes that go through this double screen with genes others have found to turn on when embryonic stem cells differentiate and we will interrogate our 400+ Agilent microarray zebrafish developmental database to identify general developmental expression patterns and genes that appear to be coregulated in time. Through this work we hope to not only test the hypothesis but to begin to identify an important subset of genes "held-back" by this RAR-corepressor mechanism