| DNA methylation guides formation of transcriptionally refractive chromatin, and is a key factor in eukaryotic gene regulation and genome defense. How DNA methylation is targeted to certain sequences in the genome is an important question. In plants, both intrinsic DNA structures and unusual RNA molecules have been proposed to be methylation signals. This thesis describes the elucidation of the methylation signal for the endogenous PAI tryptophan biosynthetic genes in Arabidopsis thaliana.;In the Arabidopsis strain Wassilewskija (WS), there are four PAI genes arranged as a tail-to-tail inverted repeat of two genes PAI1-PAI4 plus two unlinked singlet genes PAI2 and PAI3. To test the hypothesis that the PAI1-PAI4 inverted repeat locus creates a signal for PAI methylation, I investigated the natural variation in PAI gene arrangements and methylation in a collection of wild Arabidopsis isolates. Consistent with the hypothesis, I found that only strains with a PAI1-PAI4 inverted repeat had methylated PAI genes, whereas strains that carried just a single PAI1 gene at the locus had no PAI methylation. I also found that in PAI-methylated strains, the PAI1 gene is uniquely expressed due to the presence of a novel promoter in the unmethylated sequences upstream of this gene. To test whether an RNA produced by transcription from this promoter across the PAI1-PAI4 inverted repeat might be signaling PAI methylation, I suppressed expression from the promoter using transgene-targeted methylation. In the promoter-silenced plants, methylation of the singlet PAI2 and PAI3 genes was reduced, consistent with an RNA signal for PAI methylation. However, the PAI1-PAI4 locus itself retained dense methylation, suggesting that the inverted repeat DNA structure of this locus might constitute an intrinsic methylation signal independent of RNA. This model was supported by my analysis of an internal rearrangement mutant in the PAI1-PAI4 locus that displays decreased methylation on singlet PAI genes and on the inverted repeat. The rearrangement mutant analysis is consistent with a failure in methylation signaling due to both altered RNA and an altered palindromic DNA structure for the inverted repeat. |
