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.

抽象的一般理解，即DNA的甲基化已被接受，以使基因表达沉默，有关如何调节该甲基化以及其他功能（如果有）仍在进行的信息。这些问题的遗传方法产生了一系列拟南芥突变体（DDM），其中含有降低的胞嘧啶甲基化。 DDM1功能突变的丧失导致基因组中大多数重复序列的甲基化迅速丧失，但仅从低拷贝序列中甲基化缓慢丧失。 DDM1基因最近被克隆，发现编码SW12/SNF2样蛋白，将染色质动力学视为DNA甲基化的重要过程。已经创建了测试不同模型DDM1动作模型的实验计划。 特定目标是1）测试纯化的重组DDM1和DDM1相关的复合物，以依赖DNA依赖性ATPase活性，核小体重塑和染色质组装，2）研究DDM1研究DDM1与胞嘧啶甲基转移酶或其他染色质活性相关，3）与DDDM1的能力相结合。 将进行一系列了解DDM1功能丧失后果的实验。遗传分析表明，DDM1诱导的缺陷是由于未连接基因座处的稳定变体等位基因或上皮的产生。受影响的基因组区域已定位在一个DDM1诱导的矮小线（BAL）中，到包含富含亮氨酸的重复（LRR）“抗病性”基因簇的147 Kb间隔。该集群中基因表达的变化已被确定为BAL表型的可能原因。将表征与BAL变体（EPI）等位基因相关的甲基化和染色质变化。转基因实验以确定基因簇的明显是否足以引起矮人表型。从这类研究中获得的信息类型将对我们对动植物的理解和基因工程都产生至关重要的影响。