Arabidopsis DDM1: DNA Methylation and Epigenetic Stability

虽然人们普遍认为DNA甲基化有助于抑制基因表达，但关于甲基化是如何调节的以及它的其他功能（如果有的话）仍有待进一步研究。对这些问题的遗传方法已经产生了一系列含有胞嘧啶甲基化减少的拟南芥突变体（ddm）。Ddm1功能突变缺失导致基因组中大多数重复序列的甲基化快速缺失，但低拷贝序列的甲基化缺失缓慢。DDM1基因最近被克隆并发现编码SW12/ snf2样蛋白，暗示染色质动力学是DNA甲基化的一个重要过程。建立了一个实验计划来测试DDM1不同模型的作用。具体目标是1)测试纯化的重组DDM1和DDM1相关复合物的dna依赖性atp酶活性，核小体重塑和染色质组装，2)研究DDM1是否与胞嘧啶甲基转移酶或其他染色质修饰活性相关，3)比较DDM1和野生型核提取物甲基化染色质底物的能力。将进行一系列实验来了解DDM1功能丧失的后果。遗传分析表明，ddm1诱导的缺陷是由于在非连锁位点上产生了稳定的变异等位基因或表观等位基因。受影响的基因组区域已定位在一个ddm1诱导的矮化系（bal）中，间隔147 kb，包含一个富含亮氨酸的重复（LRR）“抗病”基因簇。基因表达的变化已被确定为球形表型的可能原因。甲基化和染色质变化与bal变异体（epi）等位基因将被表征。将进行转基因实验，以确定基因簇的错误表达是否足以导致矮化表型。虽然知道DNA序列对于理解生命过程的遗传控制是必要的，但这还不够。从这类研究中获得的信息将对我们对植物和动物的理解和基因工程产生至关重要的影响。

AbstractAlthough a general understanding that methylation of DNA serves to silence gene expression has been accepted, information about how that methylation is regulated and what other functions, if any, it serves is still forthcoming. A genetic approach to those questions has produced a collection of Arabidopsis thaliana mutants (ddm) that contain reduced cytosine methylation. ddm1 loss of function mutations lead to a rapid loss of methylation from most repetitive sequences in the genome, but only a slow loss of methylation from low copy sequences. The DDM1 gene has been recently cloned and found to encode a SW12/SNF2-like protein, implicating chromatin dynamics as an important process in DNA methylation. An experimental plan to test different models of DDM1 action has been created. Specific goals are to 1) test purified recombinant DDM1 and DDM1-associated complexes for DNA-dependent ATPase activity, nucleosome remodeling and chromatin assembly, 2) investigate whether DDM1 associates with cytosine methyltransferases or other chromatin modifying activities, 3) compare the ability of ddm1 and wild-type nuclear extracts to methylate chromatin substrates. A series of experiments to understand the consequences of DDM1 loss of function will be undertaken. Genetic analysis indicates that the ddm1-induced defects are due to the generation of stable variant alleles or epialleles at unlinked loci. The genomic region affected has been localized in one ddm1-induced dwarfing line (bal) to a 147 kb interval containing a leucine-rich repeat (LRR) "disease resistance" gene cluster. A change in gene expression in this cluster has been identified as a possible cause of the bal phenotype. Methylation and chromatin changes associated with bal variant (epi) alleles will be characterized. Transgenic experiments to determine if misexpression of the gene cluster is sufficient to cause the dwarfing phenotype will be done.Although knowing the DNA sequence is necessary to an understanding of genetic control of life processes, it is not sufficient. The type of information gained from studies of this sort will have critical impact upon both our understanding and genetic engineering of both plants and animals.