The role of genomic imprinting in placental metabolic function

基因组印记是一种分子过程，它区分某些基因的亲本来源，例如一个等位基因表达而另一个等位基因沉默。许多印记基因在胎盘中表达，其中一些已知在胎盘发育和代谢中起关键作用。大多数印记基因是紧密相连的簇。哺乳动物基因组中至少有16个印迹簇，每个印迹簇由差异甲基化结构域（DMD）控制。DMD的一个亲本等位基因在配子发生过程中甲基化，并且这种甲基化在受精后通过DNA胞嘧啶甲基转移酶1（DNMT1）的作用而延续。在8-细胞胚胎阶段，这种活性来自母体效应同种型DNMTIO。基因工程改造为缺乏卵母细胞衍生的DNMTIO的小鼠胚胎具有50%的正常DMD甲基化水平，并且是在不同印迹簇上缺乏DMD甲基化的细胞的表观遗传镶嵌。在没有DNMTIO的情况下，胎盘中的印记也被严重破坏。因此，DNMTIO缺陷的遗传模型是有用的，以研究基因组印迹在胎盘功能中的作用。本研究的主要目的是通过鉴定DNMTIO缺陷胎盘中的功能缺陷来确定印迹在小鼠胎盘代谢功能中的整体作用。 这一目标将在三个目标中实现。 目的1：定义DNMTIO缺陷胎盘中印迹缺陷和燃料代谢缺陷之间的因果关系。将使用RNA原位杂交、组织学和印迹基因表达和DNA甲基化的定量测量的组合来确定E9.5-E17.5 DNMTIO缺陷胎盘中印迹基因、形态和代谢异常之间的关系。我们期望从一个或多个印迹簇中发现DMD甲基化的显著变化，并将这些变化与特定的形态和代谢缺陷相关联。 目的2：确定与胎盘代谢和功能相关的新途径。这里的目标是使用各种实验技术，包括全基因组表达研究，以确定在DNMTIO缺陷胎盘中被破坏的已建立和新的代谢途径。我们期望在母胎界面的发育、脂质代谢、糖原合成、糖原生成细胞的分解和迁移中发现缺陷。 目的3：确定离散印迹基因在胎盘代谢燃料加工中的作用。 基于一些标准，将在基因敲除小鼠和过表达基因至正常水平两倍的转基因系中研究单个印记基因在胎盘代谢中的作用。

Genomic imprinting is a molecular process that distinguishes the parental origins of certain genes, such that one allele is expressed and the opposite allele is silent. Many imprinted genes are expressed in the placenta, where some are known to play critical roles in placental development and metabolism. Most imprinted genes are organized in tightly linked clusters. There are at least 16 imprinted clusters in the mammalian genome, with the imprinting of each governed by a differentially methylated domain (DMD). One parental allele of a DMD becomes methylated duhng gametogenesis, and this methylation is perpetuated after fertilization by the action of the DNA cytosine methyltransferase 1 (DNMT1) enzyme. At the 8-celt embryonic stage this activity is from the maternal-effect isoform DNMTIo. Mouse embryos genetically engineered to lack oocyte-derived DNMTIo have 50% of the normal level of DMD methylation and are epigenetic mosaics of cells lacking DMD methylation on different imprinted clusters. Imprinting in the placenta is also severely disrupted in the absence of DNMTIo. Thus, the genetic model of DNMTIo deficiency is useful to study the role of genomic imprinting in placental function. The main objective of this research is to determine the global role of imprinting in the metabolic function of the mouse placenta by identifying the functional defects in DNMTIo-deficient placentas. This objective will be pursued in three Aims. Aim 1 : Define causal relationships between defects in imprinting and fuel metabolism in DNMTIo deficient placentas. The relationship among abnormalities in imprinted genes, morphology and metabolism in E9.5-E17.5 DNMTIo-deficient placentas will be determined using a combination of RNA in situ hybridization, histology and quantitative measurements of imprinted-gene expression and DNA methylation. We expect to find significant changes in the methylation of DMDs from one or more imprinted clusters and correlate these changes with specific morphological and metabolic defects. Aim 2: Identify novel pathways relevant to placental metabolism and function. The goal here is to use a variety of experimental techniques, including genome-wide expression studies, to identify established and novel metabolic pathways that are disrupted in DNMTIo-deficient placentas. We expect to find defects in the development of the maternal-fetal interface, lipid metabolism, and glycogen synthesis, breakdown and migration of glycogen producing cells. Aim 3: Determine the role of discrete imprinted genes in processing of placental metabolic fuels. Based on a number of criteria, the roles of individual imprinted genes in placental metabolism will be studied in knockout mice and transgenic lines overexpressing a gene to twice its normal level.