Date of Graduation

8-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Srivastava, Vibha

Committee Member

Murphy, J. Brad

Second Committee Member

Pinto, Ines

Third Committee Member

Goggin, Fiona L.

Keywords

Arabidopsis thaliana; DNA methylation; Epigenetics; Phytochrome A epiallele; Transcriptional suppression

Abstract

Cytosine methylation in DNA is an integral part of epigenetically controlled regulatory networks in eukaryotes. Both plants and vertebrates display DNA methylation in the gene coding region; however, its role in gene expression is not well understood. Gene promoter, on the other hand, remains largely unmethylated. Acquisition of methylation in promoter results in transcriptional suppression of the gene. The goal of this research is to study the effect of coding region methylation in gene expression using a unique gene model, phyA'. phyA' is a transcriptionally suppressed epiallele of the Arabidopsis thaliana Phytochrome A gene, which contains methylation in CG sites resident to the exonic region. These exonic methylations confer a strong phyA mutant phenotype, characterized by elongated hypocotyls in seedlings grown under continuous far-red light (FRc). Chromatin immunoprecipitation analysis of phyA' indicated that the repressive histone mark H3K9me2 is not associated with the phyA' locus, and no significant change in the association of euchromatic mark H3K4me3 occurs. Genetic analysis ruled out the involvement of the well-known chromatin modification factors and RNAi genes in phyA' silencing, suggesting that phyA' silencing is not controlled by the RNA-mediated DNA methylation pathway. To identify novel factors involved in phyA' silencing, the forward genetics approach was taken that involved mutagenesis of the phyA' epimutant and screening for suppressor mutations. Phenotypic (primary) and molecular (secondary) screening resulted in the isolation of a suppressor line termed as suppressor of phyA' silencing 1 (sps1). Genetic and molecular analysis revealed that sps1 is a second-site mutation that reactivates the phyA' locus in spite of phyA' hypermethylation. Microarray analysis suggested that targets of SPS1 are the expressed genes. Based on the genetic and molecular data, it is proposed that the function of SPS1 is to maintain the epigenetic state of the euchromatic loci defined by their methylation state.

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