Do differentially regulated regions (DMRs) control imprinting of the maize fie1 and fie2 genes?

植物和动物早期发育的许多方面都受到遗传印记系统的控制，对于一个特定的基因，母本或父本拷贝（等位基因）被沉默。重要的是，植物中的印记只影响胚乳-在双受精后形成的两个结构之一（另一个是胚胎）。胚乳可以被认为类似于动物卵的蛋黄，为发育中的胚胎提供营养。重要的是，胚乳形成了所有谷物的大部分谷粒，因此其正确的发育（和改良）具有重要的商业和社会学意义。最近在植物上的工作，其中一些是在申请人的实验室中进行的，已经表明--再次如在动物中一样--印记等位基因的表达是通过甲基化形式的对DNA的化学修饰来调节的。我们现在已经证明，这种甲基化在玉米中一对重要的发育基因（fie 1和fie 2）的母本和父本等位基因中是不同的，而且令人惊讶的是，这种甲基化在这两个基因中以不同的方式表现，这表明它们的印记受到不同机制的调节。有趣的是，对于一个基因（fie 1），父本拷贝在配子中被甲基化，而fie 2的父本拷贝只在受精后被甲基化。来自植物的少量数据表明甲基化与基因沉默直接相关，但在动物中情况并非如此，在动物中，甲基化仅形成复杂过程的一个组成部分，通过该复杂过程，在称为DMR（差异甲基化区域）的区域中对DNA和相关蛋白质进行修饰。我们现在想剖析植物中的这种调控途径，首先确定是否是甲基化本身使基因表达沉默，然后探索植物中的DMR在DNA甲基化之前被修饰的组蛋白和其他蛋白质标记的可能性，这些蛋白质构成染色体基质（染色质）的一部分。在这项工作中，我们将使用不能进行甲基化或修饰组蛋白的植物突变株系，以及一些久经考验的转基因方法来研究个体DMR水平的变化。最后，我们将使用最近开发的染色质免疫沉淀系统（ChIP）来识别与DMR相关的蛋白质，并发现这些蛋白质中的一组，Polycomb组，是否调节自身和印迹。

Many aspects of early development in plants and animals are controlled by systems of genetic imprinting by which, for a particular gene, either the maternal or the paternal copy (allele) is silenced. Importantly, imprinting in plants affects only the endosperm - one of the two structures (the other being the embryo) formed following double fertilisation. The endosperm can be regarded as analogous to the yolk of an animal egg, providing nutrients to the developing embryo. Importantly the endosperm forms the bulk of the grain of all cereals, and its correct development (and improvement) is thus of great commercial and sociological significance. Recent work on plants, some in the applicant's laboratory, has shown - again as in animals - that expression of imprinted alleles is regulated by chemical modifications to the DNA in the form of methylation. We have now shown that this methylation differs in the maternal and paternal alleles of an important pair of developmental genes in maize (fie1 and fie2), and that, surprisingly, this methylation behaves in different ways in these two genes, suggesting that their imprinting is regulated by differing mechanisms. Interestingly, for one gene (fie1) the paternal copy is methylated in the gametes, while the paternal copy of fie2 is only becomes methylated after fertilisation. The small amount of data from plants suggest that methylation is directlty associated with gene silencing, but this is not the case in animals where it forms only one component of a complex process by which modifications are made both to the DNA and to associated proteins in regions termed DMRs (Differentially Methylated Regions). We now want to dissect this regulatory pathway in plants, first by determining whether it is the methylation itself that silences gene expression, and then by exploring the possibility that the DMRs in plants are marked, prior to DNA methylation, by modified versions of histones, and other proteins which form part of the chromosome matrix (the chromatin). For this work we shall use mutant lines of plants incapable of carrying out methylation, or modifying histones, and a number of well-tried transgenic approaches to investigate changes at the level of individual DMRs. Finally we shall use a recently-developed chromatin-immunoprecipitation system (ChIP) to identify the proteins associated with the DMRs, and to discover whether one group of these proteins, the Polycomb Group, regulate both themselves and imprinting.