GENETICS OF DNA METHYLATION PATTERNING IN ARABIDOPSIS

胞嘧啶甲基化是DNA的一种表观遗传修饰，在整个真核生物的细胞记忆中起着关键作用。它在包括亲本基因组印记、x染色体失活、转座子沉默和其他多拷贝dna在内的几个表观遗传基因调控系统中都很重要。甲基化也在肿瘤细胞生物学中发挥作用，因为肿瘤经常显示特定基因的全基因组去甲基化和超甲基化。尽管甲基化很重要，但人们对如何建立和维持适当的甲基化模式知之甚少。该研究的长期目标是利用拟南芥作为模型遗传系统来识别和研究参与适当基因组甲基化模式的基因。考虑到有能力进行大规模的诱变实验，考虑到国际上对拟南芥基因组测序的努力，考虑到甲基化突变体的收集和已经可用的克隆DNA甲基转移酶基因，拟南芥是研究这种类型的最佳生物之一。我们发现了一个可操纵的表观遗传系统，其中两个关键的花调节基因，SUPERMAN和AGAMOUS的甲基化状态可以通过影响整体基因组甲基化的几个突变而改变。令人惊讶的是，我们发现减少整体基因组甲基化的突变会导致SUPERMAN和AGAMOUS的密集高甲基化。此外，我们还发现了一类新的突变，它们也会导致SUPERMAN位点的密集过甲基化，但不会影响总体基因组甲基化水平。本课题的具体目标是：1)绘制和研究导致SUPERMAN超甲基化的突变，目标是克隆相应的基因；2)利用大规模诱变技术鉴定维持SUPERMAN密集甲基化模式的重要基因；3)研究SUPERMAN甲基化在正常发育过程中的作用；4)确定导致SUPERMAN甲基化靶向的顺式作用序列。考虑到基因组甲基化机制在植物和哺乳动物之间可能是保守的，对植物基因超甲基化的研究可能会很好地揭示动物中发生的类似过程，特别是癌细胞中基因超甲基化的表面相似现象。

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.