Genomic Imprinting in Development and Disease

与哺乳动物中表达的大多数基因不同，印迹基因仅由一个亲本等位基因表达，而哪个等位基因取决于特定的基因。因此，胰岛素样生长因子2 （Igf2）几乎完全由父系等位基因表达，而CDK抑制剂p57Kip2则由母系等位基因表达。在脊椎动物中，这种形式的基因调控是哺乳动物所特有的。它存在的原因尚不清楚。大量证据表明，印迹基因参与调节细胞增殖和生存能力。在雄激素胚胎中，整个基因组来自父系，在妊娠中期表现出胚胎外膜的过度生长和胎儿大小的增加。孤雌生殖胚胎，其全部基因组来自母体，表现出胚胎发育迟缓。为了促进对印迹的理解和鉴定新的印迹基因，我们建立了纯雄激素或孤雌生殖的成纤维细胞系。细胞系表现出完全相反的生长模式，雄激素细胞周期短，达到较高的饱和密度并形成肿瘤，而孤雌细胞衰老和死亡。利用缺乏印迹基因（如Igf2）的小鼠系，其受体之一Igf2r和p57Kip2显示，Igf2是调节这些细胞增殖和活力的主要决定因素。除了这些生长研究之外，我们还使用这些细胞系来鉴定新的印迹基因。通过抑制减法筛选，我们发现核受体辅助因子抑制/激活因子Zac1和epsilon肌糖聚糖是来自父本等位基因表达的印迹基因，以及来自母本等位基因转录的在大脑中强烈表达的Est。目前的研究主要集中在确定这些基因在发育和生长调节中的功能。我们已经开发了几种小鼠模型，用于研究一种与印痕缺陷有关的人类先天性疾病，称为普瑞德-威利综合征。在这种情况下，新生儿的特点是张力过低，呼吸模式中断，通常不能在他们生命的第一年茁壮成长。那些存活下来的人会患上饮食失调和嗜食症（摄入过多的食物），经常导致肥胖。这种疾病与父亲15号染色体部分缺失有关。在小鼠7号染色体上发现了一个同源区域。我们最近描述了在Prader-Willi地区缺乏Necdin基因父本等位基因的小鼠的衍生。这样的老鼠在出生后不久就会因呼吸系统问题而死亡，并模仿了普瑞德-威利综合征的一个方面。它们的呼吸生理学正在被更详细地研究，以确定Necdin的功能。我们还在基因靶向小鼠模型中分析了另一个与Necdin密切相关的基因Magel2的功能，该基因也来自父系等位基因，在大脑中调节昼夜节律和食欲的几个关键区域特异性表达。我们能够证明，携带被破坏的Magel2等位基因的杂合动物的父本遗传表现出严重的昼夜节律性破坏。在持续的黑暗环境中，所有的KO小鼠在短时间内都出现了严重的心律失常，而对照组野生型的幼崽在相同的环境中表现出强劲的昼夜节律。此外，我们还发现Magel2基因缺失小鼠的摄食行为被破坏，尽管与人类PWS个体相反，Magel2基因缺失小鼠的平均摄食量似乎比野生型小鼠少，并且食欲调节激素ghrelin的共线抑制。在神经解剖水平上，我们发现在Magel2缺失小鼠中，表达觉醒和食欲调节神经肽orexin的神经元数量几乎是野生型动物的两倍。总之，我们得出结论，我们的Magel2缺陷小鼠系为PWS的第二阶段病理提供了令人满意的模型。总的来说，印迹的分子分析将提供对基因表达的表观遗传控制的见解，这是一个与理解某些肿瘤抑制基因在癌症形成中的调控越来越相关的方面

Unlike the majority of genes expressed in mammals, imprinted genes are expressed from only one parental allele- which allele depends on the particular gene. Thus the Insulin like growth factor 2 (Igf2) is expressed almost exclusively from the paternal allele, whereas p57Kip2, a CDK inhibitor is expressed from the maternal allele. This form of gene regulation is, among vertebrates, unique to mammals. Why it exists is still unclear. Much evidence has suggested that imprinted genes are involved in regulating cell proliferation and viability. Androgenetic embryos, in which the entire genome is paternal in origin, exhibit overgrowth of the extraembryonic membranes and in increase in fetal size at mid gestation. Parthenogenetic embryos, where the entire genome is maternal in origin show retarded embryonic growth. To facilitate an understanding of imprinting and to identify novel imprinted genes, we established fibroblast lines which are either exclusively androgenetic or parthenogenetic in origin. The lines show diametrically opposite patterns of growth with the androgenetic cells having a shorter cell cycle time, reaching a higher saturation density and forming tumors, whereas the parthenotes senesced and died. Using mouse lines deficient for imprinted genes such as Igf2, one of its receptors, the Igf2r, and p57Kip2 revealed that Igf2 was a major determinant regulating proliferation and viability of these cells. In addition to these growth studies, we have used these lines to identify novel imprinted genes. Using a suppressive subtractive screen we identified the nuclear receptor cofactor repressor/activator Zac1 and epsilon sarcoglycan as being imprinted genes expressed from the paternal allele, as well as an Est that is strongly expressed in the brain, and is transcribed from the maternal allele. Current studies are centered on determining the function of these genes in development and growth regulation. We have developed several mouse models for a human congenital disease associated with a defect in imprinting called Prader-Willi syndrome. In this condition, newborns are featured by hypotonia, have disrupted breathing patterns and often fail to thrive past the first year of their lives. Those that survive develop an eating disorder and hyperphagia (excessive food intake) frequently resulting in obesity. The disease is associated with loss of part of the paternal chromosome 15. A homologous region is found on mouse chromosome 7. We recently described the derivation of mice lacking the paternal allele of a gene Necdin located in the Prader-Willi region. Such mice die shortly after birth due to respiratory problems and have mimicked one aspect of Prader-Willi syndrome. Their respiratory physiology is being studied in greater detail to determine the function of Necdin. We have also analyzed in gene targeting mouse model the function of another closely linked gene to Necdin, Magel2, that is also expressed from the paternal allele specifically in several key areas in the brain known to regulate the circadina rhythm and the appetite. We were able to demonstrate that the heterozygous animals bearing a disrupted Magel2 allele inherited paternally show severe disruption of circadian rhythmicity. All KO mice became severely arrhythmic within a short time period when subject to entraining in the constant darkness, whereas the control wild-type littermates displayed the robust circadian rhythm after several weeks of the same regime. We moreover were able to identify disrupted feeding behavior in Magel2 null mice, although contrary to teh human PWS individuals Magel2 KO animals appeared to consume on the average less food than their wild-type littermates, and displayed the collinear supression in the appetite-regulating hormone ghrelin. On the neuro-anatomical level we found that in Magel2 null mice the count of neurons expressing arousal and appetite regulating neuropeptide orexin is almost twice reduced as compared to the wild-type animals. In summary, we conclude that our Magel2 deficient mouse line provides a satisfactory model for the second stage of PWS pathology. Overall, a molecular analysis of imprinting will provide insights into the epigenetic control of gene expression, an aspect that is of increasing relevance to understanding the regulation of certain tumor suppressor genes in cancer formation