Date of Award

Fall 2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Horticulture and Landscape Architecture

First Advisor

Jian-Kang Zhu

Committee Chair

Jian-Kang Zhu

Committee Member 1

Ray A. Bressan

Committee Member 2

Stephen C. Weller

Committee Member 3

Larry Murdock

Abstract

DNA methylation is a key epigenetic mark for transcriptional gene silencing in many eukaryotes. DNA methylation status can be dynamically controlled by methylation and active demethylation processes. Compared to the well-known mechanisms of DNA methylation, the mechanisms of DNA demethylation and its regulation are poorly understood. In order to better understand the DNA demethylation pathway, we developed two genetic screening systems in Arabidopsis to identify new components involved in the DNA demethylation. In the first system, which is based on 35S promoter driven SUC2 (sucrose transporter 2) transgene, a mutant (91-1) was isolated and map-based cloning identified the responsible gene as MBD7(methyl-CpG binding protein 7). As important interpreters of DNA methylation, Methyl-CpG-Binding Domain (MBD) proteins are known to be involved in gene silencing through recruitment of transcriptional repressors and protein factors for heterochromatin formation. Our data show that Arabidopsis MBD7 functioins as a cellular anti-silencing factor. MBD7 is required for the expression of transgenes by preventing DNA hypermethylation at the transgene promoter. Analysis of the methylome ofmbd7 mutant plants revealed that MBD7 prevents DNA hypermethylation at thousands of genomic loci. MBD7 have been characterized previously as methyl-CpG-binding proteins in vitro, but its in vivo binding targets are not known. We performed chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to characterize the genome-wide binding targets of MBD7 in plants. We found that MBD7 preferentially binds to loci with dense and high levels of mCG. This binding is associated with the role of MBD7 in preventing the spread of DNA methylation. Protein interaction assays revealed that MBD7 is physically associated with other anti-silencing factors such as IDM1 (Increased DNA methylation 1), IDM2, and IDM3. In fact, MBD7 interacts directly with the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 (a histone H3 acetyltransferase) and IDM2 are known to function as regulatory factors for ROS1, a methylcytosine DNA glycosylase/lyase critical for active DNA demethylation. Our results suggest that MBD7 binds to genomic regions with dense and highly methylated CGs and prevents the spread of DNA methylation by recruiting other anti-silencing factors such as regulators and enzymes for active DNA demethylation. The second system is based on the RD29A promoter-drivenLUC (Luciferase) transgene. Previous studies in our lab have identified two important mutants from this system, including ros (repressor of silencing) 1and ros3. ROS1 is the first DNA demethylase identified, and ROS3 is a regulatory component in the active DNA demethylation pathway. In the current study, we carried out an enhancer mutant screen in the ros3background and isolated and characterized a mutant, rte1 (ros three enhancer 1). Our results suggest that RTE1 is a novel factor involved in the regulation of active DNA demethylation

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