Phylogenetic Comparisons of Imprinted Domains

描述(申请人提供)：基因组印记是指基因的表观遗传标记，其结果是亲本依赖的、单等位基因的表达。印记基因在胚胎生长和行为中起着关键作用；许多印记基因还在体细胞中作为癌症易感基因发挥作用，因为它们的功能单倍体状态使它们容易失活或过度表达。在印记中心的区域控制下，印记基因的位置聚集加剧了这种高度易感性，当印记中心在遗传或表观遗传学上被破坏时，会导致包括癌症在内的多基因表型异常。印迹基因还可能机械地将甲基化缺陷等营养扰动与癌症的病因直接联系起来，因为它们的顺式作用调控元件在表观遗传上是不稳定的。我们建议通过对现存的三个哺乳动物目(原始目、变形目和真兽目)成员的同源序列的系统发育比较来描述印记结构域结构的进化，并定义基本的顺式作用印记调控元件，这些元件从表观遗传上区分亲代等位基因，也构成表观遗传失调的靶标。这项拨款申请的总体假设是，基因启动子沉默代表原始印记机制，持续的亲本间遗传冲突导致一些印记区域的调控复杂性增加，因为母本和父本基因组进化出了克服基因抑制的抵消策略。因此，在更原始的哺乳动物中，印记机制被假设为不那么复杂。为了验证这一新的假设，我们最近从印迹负鼠(Didelphus Virginiana)和非印迹鸭嘴兽(Ornithorhynchus Anatinus)中建立了细菌人工染色体(BAC)文库，以检查包含三种不同印迹机制调控的癌症相关基因的印迹结构域。母体对表达的抑制将由NNAT(Neuronatin)模拟；父亲对反义转录本表达的抑制将由M6P/IGF2R模拟，而父母通过干预印迹中心对并列基因的相互抑制将由IGF2/H19和DLK1/MEG3印迹结构域模拟。我们将测试在这些比较中发现的新调控元件的功能相关性，方法是确定它们是否可以直接获得转基因小鼠的印记。这些比较系统发育研究的成功完成将极大地增强我们对这种独特的哺乳动物形式的基因调控的进化的理解。这些研究对于识别新的印记基因，以及确定已知含有与癌症和神经遗传性疾病(如精神分裂症、双相情感障碍和自闭症)机械相关的遗传和/或表观遗传突变的定义较少的印记区域也将是至关重要的。

DESCRIPTION (provided by applicant): Genomic imprinting refers to an epigenetic marking of genes that results in parent-of-origin dependent, monoallelic expression. Imprinted genes have critical roles in embryonic growth and behavior; many also function as cancer susceptibility loci in somatic cells because their functionally haploid state makes them vulnerable to inactivation or overexpression. This heightened susceptibility is exacerbated by positional clustering of imprinted genes under the regional control of imprinting centers that, when disrupted genetically or epigenetically, lead to multigenetic phenotypic abnormalities including cancer. Imprinted genes may also mechanistically link nutritional perturbations like methylation deficiency directly to the etiology of cancer because their cis-acting regulatory elements are epigenetically labile. We propose to use phylogenetic comparisons of orthologous sequences from members of the three extant mammalian orders, Prototheria, Metatheria and Eutheria to characterize the evolution of imprinted domain structures and define the fundamental cis-acting imprint regulatory elements that epigenetically distinguish the parental alleles and also constitute targets for epigenetic dysregulation. The overall hypothesis of this grant application is that gene promoter silencing represents the primordial imprint mechanism, and that ongoing interparental genetic conflict has led to increased regulatory complexity for some imprinted domains, as the maternal and paternal genomes evolved counteracting strategies to overcome gene repression. Imprinting mechanisms are therefore postulated to be less complex in more ancestral mammals. To test this novel hypothesis we recently produced bacterial artificial chromosome (BAC) libraries from the imprinted opossum (Didelphus virginiana) and the non-imprinted platypus (Ornithorhynchus anatinus) to examine imprinted domains containing genes involved in cancer that are regulated by three different imprinting mechanisms. Maternal repression of expression will be modeled by NNAT (Neuronatin); paternal repression of expression with antisense transcripts will be modeled by M6P/IGF2R, and reciprocal parental repression of juxtapositioned genes by an intervening imprint center will be modeled by the IGF2/H19 and DLK1/MEG3 imprinted domains. We will test the functional relevance of novel regulatory elements identified in these comparisons by determining if they can direct acquisition of imprinting in transgenic mice. The successful completion of these comparative phylogenetic studies will significantly enhance our understanding of the evolution of this unique mammalian form of gene regulation. These studies will also be critical for identifying novel imprinted genes, and characterizing the less well-defined imprinted domains known to harbor genetic and/or epigenetic mutations mechanistically involved in cancer and neurogenetic disorders, such as schizophrenia, bipolar disease and autism.