Phase I enzymes as part of an epigenetic switch

描述(申请人提供)：这一建议是基于这样的假设，即第一阶段的酶参与胚胎开关，并且在受精时开关的默认位置是“关”。这将发生在其调控DNA中含有维甲酸反应元件(RARE)的基因子集中。“关”的位置可能是由于维甲酸受体的一种功能，但还没有得到足够的注意：在维甲酸的缺失中，辅阻遏物分子与RAR？在稀有土地上，岩心抑制器阻止了RAR？通过与辅活化子的相互作用，还引入了一种浓缩染色质的组蛋白去乙酰基酶。随着研究的进行，维甲酸通过乙醛脱氢酶被精确地解析出来，当它离开细胞来源时，维甲酸可以进入邻近的细胞，但受到表达特定细胞色素P450活性的细胞边界的限制(cyp26a1、cyp26b1、cyp26c1)，后者将维甲酸氧化成不起作用的配体。所有这些成分在发育中的胚胎中都受到仔细的调节，并有受维甲酸影响的直接和/或间接反馈回路。维甲酸的分配会降低基因的表达，影响胚胎细胞的分化。那么，这意味着什么？为什么它很重要？在成人中被认为是功能I相酶的基因在胚胎的发育中起着关键作用-药物；影响这些基因或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。我们将使用反义吗啉、指示性转基因胚胎和额外的试剂和方法来机械地检验这一假说，以确定被抑制的基因，并看看对这些成分的实验操作是否会对生殖系产生跨代影响。具体地说，我们将使用转基因维甲酸指示剂斑马鱼胚胎(在本实验室开发)来帮助我们确定在合子基因表达启动时，哪些基因可能通过这种机制被抑制。我们将通过使用抗Smrt抗血清免疫沉淀斑马鱼Smrt辅抑制子相关染色质来证实这种联系。我们将通过双重筛选将已识别的基因家族与其他已发现的在胚胎干细胞分化时启动的基因进行比较，并询问我们的400+Agilent微阵列斑马鱼发育数据库，以确定一般的发育表达模式和似乎在时间上共调控的基因。通过这项工作，我们希望不仅检验这一假说，而且开始识别通过这种RAR-辅阻遏子机制“抑制”的一个重要基因子集

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