Arabidopsis DDM1: DNA Methylation and Epigenetic Stability

拟南芥 DDM1：DNA 甲基化和表观遗传稳定性

• 批准号: 9985348
• 负责人: Eric Richards
• 金额: $ 46.5万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9985348&HistoricalAwards=false
• 关键词: Arabidopsis; DDM1; DNA; Methylation; Epigenetic

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.

虽然DNA甲基化作用于沉默基因表达的一般认识已经被接受，但关于甲基化是如何被调节的以及它所起的其他作用（如果有的话）的信息仍然是即将到来的。针对这些问题的遗传学方法已经产生了一系列含有减少的胞嘧啶甲基化的拟南芥突变体（ddm）。ddm 1功能丧失突变导致基因组中大部分重复序列的甲基化快速丧失，但仅导致低拷贝序列的甲基化缓慢丧失。 DDM 1基因最近被克隆并发现编码SW 12/SNF 2样蛋白，暗示染色质动力学是DNA甲基化的重要过程。一个实验计划，以测试不同的模型DDM 1的行动已经创建。 具体目标是：1）检测纯化的重组DDM 1和DDM 1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DDM 1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ddm 1和野生型核提取物甲基化染色质底物的能力。 将进行一系列实验以了解DDM 1功能丧失的后果。遗传分析表明，ddm 1诱导的缺陷是由于在非连锁位点产生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被定位在一个ddm 1诱导的矮化系（bal）的147 kb的间隔含有富含亮氨酸的重复序列（LRR）的“抗病”基因簇。该簇中基因表达的变化已被鉴定为bal表型的可能原因。与bal变体（epi）等位基因相关的甲基化和染色质变化将被表征。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。尽管知道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类型将对我们对植物和动物的理解和基因工程产生关键影响。