Genetic Analysis of H19 and Igf2 Imprinting Regulation

基因组印记是一种表观遗传机制，影响哺乳动物中的一小部分基因，并导致单等位基因的亲本特异性表达。许多印迹基因编码着增殖或分化的调节器，因此它们调节的变化可能在发育异常和癌症中发挥作用。因此，全面了解印迹的调控机制对于理解正常的胚胎发育和许多人类疾病的发病机制至关重要。H19和Igf2是两个连锁基因，具有相似的时间和组织特异性表达模式，并被印记。H19在母系等位基因中表达，而Igf2在父系中表达。这些基因座印记的协调调节依赖于位于H19基因上游的2kb差异甲基化区域(DMD)。这个DMD包含四个富含GC的重复序列，它们在小鼠、大鼠和人类序列之间高度保守，这一事实表明它们是该结构域调控的重要特征。这四个重复序列在体外表现出甲基化依赖的边界活性。这些研究的第一个目标是确定在DMD内通过重复四个富含CG的21个重复来建立印记的要求。第二个目的是确定这些重复序列中特定CG二核苷酸的甲基化是否是等位基因亲本同一性的信号。这两个目标都将通过以内源基因为靶点来实现。这些实验将有助于加深对印迹形成的分子机制的理解。

Genomic imprinting is an epigenetic mechanism affecting a small group of genes in mammals and that results in monoallelic parental-specific expression. Many imprinted genes code for regulators of proliferation or differentiation, so alterations in their regulation can play a role in developmental abnormalities and cancer. Therefore, gaining a full comprehension of the mechanisms by which imprinting is regulated is crucial to understanding normal embryonic development and the pathogenesis of many human diseases. H19 and Igf2 are two linked genes with a similar temporal and tissue- specific pattern of expression and are imprinted. Hl9 is expressed from the maternal allele, whereas Igf2 is expressed paternally. The coordinate regulation of imprinting at these loci depends on a 2 kb differentially methylated domain (DMD) that lies upstream of the H19 gene. This DMD contains four GC-rich repeats that are highly conserved between the mouse, rat and human sequences, a fact that suggests that they are an important feature for the regulation of the domain. The four repeats exhibit methylation-dependent boundary activity in vitro. The first goal of these studies is to determine the requirement of the four CG-rich 21- by repeats within the DMD for the establishment of imprinting. The second aim is to determine whether methylation of specific CG dinucleotides within these repeats is a signal for the parental identity of the alleles. Both aims will be accomplished by gene targeting at the endogenous locus. These experiments will lead to enhanced understanding of the molecular mechanisms underlying imprinting.