Phylogenetic Comparisons of Imprinted Domains

描述（由申请人提供）：基因组印记是指基因的表观遗传标记，导致依赖于父母来源的单等位基因表达。印迹基因在胚胎生长和行为中起关键作用；许多基因在体细胞中也有癌症易感位点的功能，因为它们的功能单倍体状态使它们容易失活或过度表达。在印迹中心的区域控制下，印迹基因的位置聚类加剧了这种易感性，当印迹中心在遗传或表观遗传上受到破坏时，会导致包括癌症在内的多遗传表型异常。由于其顺式调控元件在表观遗传上是不稳定的，因此印迹基因也可能将甲基化缺乏等营养扰动直接与癌症病因学联系起来。我们建议通过对原兽目、后兽目和真兽目三种现存哺乳动物的同源序列进行系统发育比较，以表征印迹结构域结构的进化，并定义基本的顺式作用的印迹调控元件，这些元件在表观遗传上区分亲本等位基因，也构成表观遗传失调的靶点。本基金申请的总体假设是，基因启动子沉默代表了原始印记机制，并且持续的亲代遗传冲突导致一些印记域的调控复杂性增加，因为母亲和父亲的基因组进化出对抗策略来克服基因抑制。因此，印迹机制在更古老的哺乳动物中被认为不那么复杂。为了验证这一新的假设，我们最近从印迹负鼠（Didelphus virginia）和非印迹鸭嘴兽（Ornithorhynchus anatinus）中提取了细菌人工染色体（BAC）文库，以检测包含由三种不同印迹机制调节的与癌症有关的基因的印迹结构域。母源的表达抑制将通过NNAT （Neuronatin）来模拟；父本对反义转录本表达的抑制将通过M6P/IGF2R来模拟，亲本通过干预印记中心对并置基因的相互抑制将通过IGF2/H19和DLK1/MEG3印记域来模拟。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，通过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大大提高我们对这种独特的哺乳动物基因调控形式的进化的理解。这些研究对于识别新的印迹基因和描述不太明确的印迹结构域也将是至关重要的，这些印迹结构域已知包含与癌症和神经遗传疾病（如精神分裂症、双相情感障碍和自闭症）机制相关的遗传和/或表观遗传突变。

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.