一个C2H2-type锌指蛋白SUF4调控拟南芥基因组DNA甲基化模式的分子机理

DNA去甲化是DNA甲基化的相反过程, 它能维持基因处于活跃表达状态并激活已经沉默的基因。前期工作中，申请人通过遗传筛选鉴定到一个新的与DNA去甲基化有关的突变体suf4-4。SUF4突变造成基因组3842个位点发生超甲基化，并且部分超甲基化位点与EDM2,IBM1和ROS1的目标作用位点相重合。SUF4编码一个包含C2H2-type 锌指结构域的蛋白，它参与了调控IBM1基因3'的表达。在此基础上，本研究将利用Y1H,EMSA,ChIP等方法研究SUF4在35S启动子上和IBM1内含子异染色质上的结合情况,及SUF4突变对ROS1功能和EDM2调控IBM1 3'表达的影响。用Y2H,LCI,Co-IP等方法探索SUF4与EDM2和IBM2/ASI1的关系。通过上述研究，将深入理解SUF4在ROS1介导的DNA去甲基化过程中的作用及它对IBM1 pre-mRNA剪切加工的影响。

Epigenetic changes caused by DNA methylation, histone modifications and histone variants play crucial roles in the regulation of chromatin structure, genome stability, genomic imprinting and transcriptional silencing of transgenes as well as other foreign DNA. However, DNA methylation is reversible via DNA demethylation which is important for maintaining proper gene expression and activating silenced genes. Our previous genetic screening identified a new Arabidopsis mutant which carried a lesion on SUF4 gene and exhibited obviously elevated DNA methylation in many loci, and therefore it was named as suf4-4. Mutation of SUF4 led to 3842 hypermethylated loci in genome and a large proportion of these loci overlapped with those hypermethylated loci in edm2, ibm1 and ros1.SUF4 encodes a C2H2-type zinc finger protein and is involved in 3' expression of IBM1 gene. In this project, we are going to use Y1H, EMSA and ChIP approaches to study specific binding of SUF4 on 35S promoter and IBM1 intronic heterochromatin, and effects of SUF4 mutation on ROS1 functions and regulation of IBM1 3' expression by EDM2. Furthermore,Y2H, LCI and Co-IP approaches are going to be used to find interactions among SUF4, EDM2 and IBM2/ASI1. All the above studies will contribute to our better understanding of the roles of SUF4 in ROS1-mediated DNA demethylation and IBM1 pre-mRNA processing.