GENETICS OF DNA METHYLATION PATTERNING IN ARABIDOPSIS

胞嘧啶甲基化是DNA的一种表观遗传修饰，在真核生物的细胞记忆中起着关键作用。它在几个表观遗传基因调控系统中是重要的，包括亲本基因组印记，X染色体失活，转座子和其他多拷贝DNA的沉默。甲基化也在肿瘤细胞生物学中发挥作用，因为肿瘤通常表现为全基因组范围的去甲基化和特定基因的高甲基化。尽管它很重要，但人们对如何建立和维持适当的甲基化模式知之甚少。这项拟议研究的长期目标是使用拟南芥作为一个模型遗传系统来识别和研究与适当的基因组甲基化模式有关的基因。拟南芥是这类研究的最佳生物之一，因为它有能力进行大规模的突变实验，考虑到国际上对拟南芥基因组测序的努力，以及已经可用的甲基化突变体和克隆的DNA甲基转移酶基因的集合。我们发现了一个可操作的表观遗传系统，在这个系统中，两个关键的花调控基因Superman和AGAMOUS的甲基化状态可以通过几个影响整个基因组甲基化的突变来改变。令人惊讶的是，我们发现减少基因组整体甲基化的突变会导致超人和AGAMOUS的密集超甲基化。此外，我们还发现了一类新的突变，这种突变也会导致超人基因的密集过度甲基化，但不会影响整个基因组的甲基化水平。这项建议的具体目标是1)定位和研究导致超人超甲基化的突变，目的是克隆相应的基因，2)使用大规模突变来确定对维持超人密集甲基化模式至关重要的基因，3)研究超人甲基化在正常发育过程中的作用，以及4)确定导致超人靶向甲基化的顺式作用序列。考虑到基因组甲基化机制在植物和哺乳动物之间是保守的，对植物基因高甲基化的研究很可能有助于揭示动物中发生的类似过程，特别是表面上相似的癌症细胞基因高甲基化现象。

Cytosine methylation is an epigenetic modification of DNA that plays a key role in cellular memory throughout the eukaryotes. It is important in several epigenetic gene regulatory systems including parental genomic imprinting, X-chromosome inactivation, and the silencing of transposons and other multiple copy DNAs. Methylation also plays a role in tumor cell biology, as tumors often show both genome wide demethylation and hypermethylation of specific genes. Despite its importance, little is known about how proper methylation patterns are established and maintained. The long-term goal of the proposed research is to use Arabidopsis thaliana as a model genetic system to identify and study genes involved in proper genomic methylation patterning. Arabidopsis is one of the best organisms for studies of this type, given the ability to do large-scale mutagenesis experiments, given the international Arabidopsis genome sequencing efforts, and given the collection of methylation mutants and cloned DNA methyltransferase genes which are already available. We have discovered a manipulable epigenetic system in which the methylation state of two key floral regulatory genes, SUPERMAN and AGAMOUS, can be altered by several mutations that affect overall genomic methylation. Surprisingly, we find that mutations that reduce overall genomic methylation, cause dense hypermethylation of SUPERMAN and AGAMOUS. Furthermore, we have discovered a new class of mutations that also cause dense overmethylation of the SUPERMAN locus, but that do not affect overall genomic methylation levels. The specific objectives of this proposal are 1) to map and study mutations which cause hypermethylation of SUPERMAN, with the goal of cloning the corresponding genes, 2) to use large scale mutagenesis to identify genes that are important in maintaining the dense patterns of methylation at SUPERMAN, 3) to study the role of SUPERMAN methylation during normal development, and 4) to determine cis acting sequences which cause targeting of SUPERMAN for methylation. Given the likelihood that genomic methylation mechanisms are conserved between plants and mammals, studies of plant gene hypermethylation may well shed light on similar processes occurring in animals, especially the superficially similar phenomenon of hypermethylation of genes in cancerous cells.