Role of DNA methylation in imprint maintenance in differentiated human cells

基因组印记现象是许多儿童疾病综合征的核心，包括Beckwith-Weidemann综合征(BWS)、Prader-Willi综合征(PWS)和一过性新生儿糖尿病(TNDM)I型等疾病。这些综合征中的每一种都涉及一个印记基因，也就是说，通常只有两条染色体中的一条具有活性，要么是通过精子从父亲那里继承的，要么是通过卵子从母亲那里继承的。因此，这些基因会“印记”它们来自哪个父母的记忆。患有印记疾病综合征的患者在晚年患糖尿病(TNDM)或肾母细胞瘤(BWS)等进一步问题的风险也会增加。一个基因的一个拷贝失活，而它在同一细胞核中的相同伙伴保持活跃的机制仍在积极研究中。我们知道，添加到DNA上的化学标签称为DNA甲基化，与标记印记基因的非活跃拷贝有关，但尚不清楚这是否在所有情况下都足以关闭基因，或者与DNA相关的蛋白质是否更重要，这些蛋白质被称为组蛋白。虽然这样的机制问题可以在小鼠等实验动物身上得到解决，但人类研究要么使用了来自患者的细胞，在患者细胞中无法分辨DNA甲基化或组蛋白的变化是否是病因，要么使用药物操纵了癌细胞的DNA甲基化水平，这些癌细胞在遗传上不稳定，因此是一个糟糕的模型。我们已经开发出了非癌症、遗传稳定、印迹正常的人类细胞系，然后选择性地降低了DNA甲基化水平。这些细胞似乎表现出导致BWS和PWS的基因印记丢失，很可能是所有其他印记区域，但在非印记区域不受影响。它们改变了生长特征和外观，符合印记基因在调节生长和营养方面具有重要作用的理论。因此，它们代表了一种独特的资源，用于探索DNA甲基化与组蛋白状态在调节印记基因中的重要性，并通过与BWS患者和其他人的细胞进行交叉比较，应该可以确定哪一种更有可能是病因。这些细胞是一种潜在的有价值的工具，可以用来确定目前临床上使用的DNA甲基化药物的副作用。我们还可以使用这些细胞来寻找以前未被识别的印记基因，这可能与其他疾病有关。此外，我们还可以使用这些细胞进行进一步的机械性工作，包括试图确定建立印记所涉及的因素，这些印记反过来将成为这些疾病的病原体的候选者。

SummaryThe phenomenon of genomic imprinting lies at the heart of a significant number of childhood disease syndromes, including disorders such as Beckwith-Weidemann Syndrome (BWS), Prader-Willi Syndrome (PWS) and Transient neonatal diabetes mellitus (TNDM) type I. Each of these syndromes involves a gene which is imprinted, that is to say is normally only active from one of the two chromosomes in the cell, either the one inherited from the father via the sperm, or the one inherited from the mother via the egg, the genes thus being "imprinted" with a memory of which parent they came from. Patients with imprinted disease syndromes have increased risks for further problems such as diabetes (TNDM) or Wilms' tumour (BWS) in later life as well. The mechanism by which one copy of a gene becomes inactivated, while its identical partner in the same cell nucleus remains active, is still under active research. We know that chemical tags added to the DNA, called DNA methylation, are involved in marking the inactive copy of the imprinted gene, but it is not known whether this is sufficient in all cases to turn off the gene, or whether the proteins associated with the DNA, called histones, are more important. While such mechanistic questions can be addressed in experimental animals such as mice, human studies have either used cells from patients, where it is not possible to tell if the changes seen in the DNA methylation or the histones are causative, or have manipulated DNA methylation levels using drugs in cancer cell lines, which are not genetically stable and thus a poor model. We have developed human cell lines which are non-cancerous and genetically stable, with normal imprinting, and then selectively reduced the levels of DNA methylation only. These cells appear to show loss of imprinting of the genes which cause BWS and PWS and most likely all other imprinted regions, but are unaffected at non-imprinted regions. They have altered growth characteristics and appearance, in line with theories that imprinted genes have important roles in regulating growth and nutrition. They thus represent a unique resource for exploring the importance of DNA methylation versus histone status in regulating imprinted genes and by cross-comparing to cells from BWS patients and others, it should be possible to establish which is more likely to be causative. The cells are a potentially valuable tool for determining the side-effects of pharmacological inhibitors of DNA methylation currently being used in the clinic. We can also use the cells to look for previously unrecognised imprinted genes, which may be linked to additional disorders. Additionally we can use the cells to carry out further mechanistic work, including attempting to identify the factors involved in establishing imprints, which would in turn be candidates for causative agents in these diseases.