Phase I enzymes as part of an epigenetic switch

描述（由申请人提供）：该提案基于第一阶段酶参与胚胎开关的假设，并且在受精时开关的默认位置为“关闭”。这将发生在其调控DNA中具有视黄酸反应元件（RAREs）的基因子集中。“关闭”的位置可能是由于维甲酸受体的功能没有得到足够的注意：在缺乏维甲酸的情况下，辅抑制因子分子与RAR？在RAR上，协代表子阻止RAR？通过与辅激活子相互作用同时引入组蛋白去乙酰化酶使染色质凝聚。随着发育的进行，维甲酸通过醛脱氢酶被精确地分解，当维甲酸离开其细胞源时，它可以进入邻近细胞，但受到表达特定细胞色素p450活性（cyp26a1, cy26b1, cyp26c1）的细胞边界的限制，这种活性将维甲酸氧化为无功能配体。所有这些成分都在发育中的胚胎中受到精心调节，并具有受视黄酸影响的直接和/或间接反馈回路。然后，维甲酸的分配会抑制基因并影响胚胎细胞的分化。那么这意味着什么，为什么它很重要呢？在成人中被认为是功能性I期酶的基因在胚胎药物的发展中起着至关重要的作用；影响这些基因或被设计成与它们相互作用的激素可能在不知情的情况下在胚胎的发育中发挥戏剧性或微妙的作用，特别是在神经系统中。我们将利用反义morpholinos工具箱、转基因指示胚胎、其他试剂和方法来验证这一假说，以确定被抑制的基因，并观察对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器斑马鱼胚胎（在本实验室开发）来帮助我们确定在合子基因表达开始时，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共抑制因子相关染色质来证实这种关联。我们将通过这种双重筛选将已识别的基因家族与其他人发现的胚胎干细胞分化时开启的基因进行比较，我们将查询我们的400多个安捷伦微阵列斑马鱼发育数据库，以确定一般的发育表达模式和基因，这些基因似乎是同步调节的。通过这项工作，我们希望不仅能验证这一假设，还能开始识别被这种rar协同抑制机制“抑制”的重要基因子集

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