Imprinting studies of a paternally expressed Usp29 gene

描述(申请人提供)：项目摘要基因组印记与人类遗传疾病密切相关，因为它的功能半合子。人类染色体19q13.4/小鼠7号染色体近端的Peg3结构域也与一些印迹相关的遗传疾病有关，包括在胶质瘤、乳腺癌和卵巢癌患者中经常丢失人类Peg3的表达。因此，该项目的长期目标是了解控制Peg3结构域印迹的机制。在上一个资金周期中，我们预测Peg3结构域可能受一个潜在的ICR(印迹控制区)控制，即Peg3-DMR(差异甲基化区域)。我们随后证明了PEG3-DMR的缺失对PEG3结构域的转录具有全球影响。父本和母本传递这种靶向缺失也分别导致小鼠生长速度下降和增加，这是一种与小鼠近端7号染色体相关的印记表型。我们还发现，这种潜在的ICR具有非常不寻常的YY1结合位点串联阵列，并且在卵子发生期间降低体内YY1蛋白的水平导致Peg3-DMR上靶标特异性DNA低甲基化。基于这些观察，我们假设1)Peg3结构域的印迹由Peg3-DMR控制，2)这种控制需要YY1作为主要的反式因子，在卵子发生过程中靶向从头DNA甲基化。在目前的提案中，我们将通过以下三个目标来检验这些假设。AIM1将利用缺乏PEG3-DMR的突变小鼠进一步表征PEG3-DMR所扮演的ICR角色。AIM2将通过产生另一个带有突变的YY1位点的KO等位基因来剖析YY1对观察到的PEG3-DMR的ICR角色的确切贡献。AIM3将研究YY1建立和维持PEG3-DMR的等位基因特异性DNA甲基化的潜在机制。这些实验的结果将为之前未被注意到的YY1在基因组印记中的作用以及与Peg3结构域相关的人类遗传疾病提供有价值的见解。 公共卫生相关性：由于一种不寻常的剂量控制机制，即基因组印迹，少数哺乳动物基因(少于200个)在两个亲本等位基因之间功能不相等。目前的提案旨在描述基因组印迹的调控机制。

DESCRIPTION (provided by applicant): Project Summary Genomic imprinting is closely associated with human genetic disorders due to its functional hemizygosity. The Peg3 domain on human chromosome 19q13.4/proximal mouse chromosome 7 is also associated with several imprinting-related genetic disorders, including frequent loss of human PEG3 expression in the patients of glioma, breast and ovarian cancers. Thus, the long-term objective of this project is to understand the mechanisms controlling the imprinting of the Peg3 domain. In the previous funding cycle, we predicted that the Peg3 domain is likely controlled by one potential ICR (Imprinting Control Region), the Peg3-DMR (Differentially Methylated Region). We subsequently demonstrated that deletion of the Peg3-DMR has a global impact on the transcription of the Peg3 domain. Paternal and maternal transmission of this targeted deletion also resulted in decreased and increased growth rates of the mouse, respectively, an imprinting phenotype associated with proximal mouse chromosome 7. We also discovered that this potential ICR has a very unusual tandem array of YY1 binding sites, and that reducing the in vivo levels of YY1 protein during oogenesis resulted in target-specific DNA hypomethylation on the Peg3-DMR. Based on these observations, we hypothesize 1) that the imprinting of the Peg3 domain is controlled by the Peg3-DMR and 2) that this control requires YY1 as a main trans factor for targeting de novo DNA methylation during oogenesis. In the current proposal, we will test these hypotheses with the following three aims. Aim1 will further characterize the ICR roles played by the Peg3-DMR using the mutant mice lacking the Peg3-DMR. Aim2 will dissect the exact contributions of YY1 to the observed ICR roles of the Peg3-DMR by generating another KO allele with mutated YY1 sites. Aim3 will investigate potential mechanisms by which YY1 establishes and maintains the allele-specific DNA methylation of the Peg3-DMR. The results from these experiments will provide valuable insights regarding previously unnoticed roles for YY1 in genomic imprinting as well as human genetic disorders linked to the Peg3 domain. PUBLIC HEALTH RELEVANCE: A small number of mammalian genes (less than 200) are not functionally equal between the two parental alleles due to an unusual dosage control mechanism, genomic imprinting. The current proposal is aiming to characterize regulatory mechanisms of genomic imprinting.