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The Mechanisms Of LOW IN LUCIFERASE EXPRESSION Suppressing Cytosine Methylation And Transcriptional Gene Silencing In Arabidopsis Thallana

Posted on:2014-10-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:D M LiFull Text:PDF
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Cytosine methylation is crucial epigenetic modification. Some molecular and genetic studies showed that keeping the genome wide dynamic equilibrium of cytosine methylation is very important for diverse biological processes, including transposon silencing, imprinting gene expression and plant defending. While the positive feedback RdDM (RNA directed DNA Methylation) help establishing the cytosine methylation, mechanisms that counteract hypermethylation are equally important but are less well understood. To invesitage the molecular mechanism for establishing de novo methylation and transcriptional gene silencing, we designed two DNA methylation characteristic report systems (LUCH and YJ). From forward genetic screening, the gene LOW IN LUCIFERASE EXPRESSION (LIL), which encodes a HEAT SHOCK PROTEIN20(HSP20) homolog, was identified as reducing the LUC expression in the mutant. To uncover the molecular mechanisms of this phenotype, biochemistry, molecular biology and genetic study were introduced to evaluate the specificity of LIL regulation and mechanisms. To invesigate the genome-wide impact of LIL on DNA methylation and relationship between LIL and RdDM, BS-seq and small RNA libry was performed. Cell biology was introducted to study the impact of LIL on the subnuclear localization of AGO4. To better understand the molecular functions of LIL, we performed a yeast two-hybrid screening to identify LIL-interacting proteins. The results showed:(1) LIL specifically affects gene expression in RdDM loci;(2) this regulation activity is DNA methylation dependent, and the role of LIL in the promotion of LUC and endogeneous genes expression was totally alleviated when the genes lacked DNA methylation, so this is consistent with the notion that LIL acts as a negative factor in RdDM or acts downstream of RdDM to promote the expression of RdDM target genes;(3) LIL mutant increased CG and CHH methylation and70%of those regulated loci which closely associated with small RNA generated regions were found to occur in Pol IV or Pol V;(4) LIL does not impact siRNA biogenesis or the production of Pol V-dependent transcripts, and specifically affact subnuclear localization of AGO4, multiple prominent AGO4dots were present, with some apparently being close to the surface of the nucleolu, the AGO4nucleolar dot was not present, and instead, one large AGO4aggregate scattered AGO4signal;(5) LIL was found to interact with three MBD proteins: MBD5, MBD6and MBD7. These interactions affect transcriptional gene silencing at several specific loci. Get together all of our results, LIL functioned downstream of RdDM and repressed transcriptional gene silecing by regulation of genome wide DNA methylation level. In this paper, the first reported negative mechanism maintains the dynamic equilibrium of whole genome DNA methylation and gene expression pattern. The molecular mechanisms for negative regulating TGS need future study.
Keywords/Search Tags:DNA methylation, negative regulate RdDM, whole genome libryestablishment and analysis, AGO4localization
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