Analysis of the Mouse PWS Imprinting Center

Prader-Willi/Angelman综合征（PWS/AS）结构域包含一组受二分印记中心（Ie）协调调控的印记基因。PWS-IC的功能是在整个结构域中建立和维持父系表观基因型。在该域中建立和维持印记的机制以及PWS-IC功能的机制还不清楚。小鼠染色体7 C上的~ 3.8Mb同线印迹区域用作PWS和PWS-IC功能的模型。本研究的目的是探讨小鼠PWS-IC功能的机制。基于DNA酶超敏性和染色体构象捕获（3C）的研究，我们提出PWS-IC是由多个调控区域，功能组合在父系遗传的染色体上，并从事在域内的远程相互作用，以促进印记和激活基因表达的父系（帕特）染色体。我们进一步假设，帕特染色体上的印记涉及的因素，结合到PWS-IC和位点，与PWS-IC相互作用。因此，我们提出了2个具体目的：I）我们将使用3C和4C测定来全面分析涉及不同细胞类型中的母体（mat）和pat染色体上的PWS-IC的长程相互作用。这应该定义了mat和pat染色体上ASIPWS结构域的空间组织，确定了小鼠基因组中与PWS-IC相互作用的区域（可能参与印记过程），并揭示了PWS-IC的组织特异性功能。这也将使我们能够确定PWS-IC与其靶基因之间的长程相互作用是否通过循环或跟踪机制发生。2)我们将对pat染色体上PWS-IC内鉴定的6个DNase I超敏（DH）位点进行分子表征;我们假设这些DR位点是PWS-IC的活性成分，并共同构成功能性PWS-IC。我们还假设这些DH位点内的因子结合促进PWS-IC的长程相互作用并介导其功能。因此，我们将使用各种分子策略来表征位于这些DH位点内的顺式和反式作用元件。这些研究应该对PWS-IC功能产生新的见解，并为未来对其调节成分及其相互作用位点的遗传分析提供强有力的基础。

The -2 Mb Prader-Willi/Angelman syndrome (PWS/AS) domain encompasses a group of imprinted genes that are coordinately regulated by a bipartite imprinting center (Ie). The PWS-IC functions to establish and maintain the paternal epigenotype across the domain. The mechanisms by which imprinting is established and maintained in the domain, as well as mechanisms of PWS-IC function, are not weIl-nnderstood. The -3,8 Mb syntenic imprinted region on mouse chromosome 7C serves as a model for PWS and PWS-IC function. The goal of this project is to investigate mechanisms of murine PWS-IC function. Based on DNase hypersensitivity and chromosome conformation capture (3C) studies, we propose that the PWS-IC is composed of multiple regulatory regions that function combinatorially on the paternally-inherited chromosome and engage in long-range interactions within the domain to facilitate imprinting and activate gene expression on the paternal (pat) chromosome. We further postulate that imprinting on the pat chromosome involves factors that bind to the PWS-IC and to loci that interact with the PWS-IC. Thus, we propose 2 Specific Aims: I) We will use 3C and 4C assays for a comprehensive analysis oflong-range interactions involving the PWS-IC on the maternal (mat) and pat chromosomes in different cell types. This should define the spatial organization of the ASIPWS domain on the mat and pat chromosomes, identify regions across the mouse genome that interact with the PWS-IC (and may be involved in the imprinting process), and reveal tissue-specific functions ofthe PWS-IC. This will also allow us to determine if long-range interactions between the PWS-IC and its target genes occur via a looping or tracking mechanism. 2) We will perform a molecular characterization ofthe 6 DNase I hypersensitive (DH) sites we have identified within the PWS-IC on the pat chromosome; we hypothesize these DR sites are the active components of the PWS-IC and together constitute the functional PWS-IC. We also postulate that factors binding within these DH sites facilitate long-range interactions of the PWS-IC and mediate its function. Therefore, we will use various molecular strategies to characterize cis- and trans-acting elements located within these DH sites. These studies should yield new insight into PWS-IC function and provide a strong basis for future genetic analyses of its regulatory components and the sites that interact with it.