| DNA methylation and heterochromatin assembly play critical roles in gene regulation and genome stability in mammals and plants. In plants, heterochromatin formation is directed by double-stranded (ds) RNA that triggers both histone H3 lysine 9 methylation (H3 mK9) and cytosine methylation on identical DNA sequences. In the laboratory plant Arabidopsis thaliana, a previous genetic screen for mutations that confer reduced cytosine methylation identified the SUVH4 H3 K9 methyltransferase (MTase) and the CMT3 cytosine MTase. This finding indicated that SUVH4-mediated H3 mK9 is required for CMT3-mediated DNA methylation. However, suvh4 mutations confer only a partial loss of DNA methylation relative to cmt3 mutations, suggesting that additional H3 K9 MTases contribute to the CMT3 DNA methylation pathway. This thesis describes the characterization of the SUVH5 and SUVH6 MTases that work together with SUVH4 to maintain CMT3-mediated DNA methylation. Like SUVH4, SUVH5 and SUVH6 methylate H3 K9 in vitro. Additionally, in a suvh4 mutant background the suvh5 and suvh6 mutations each cause reduced residual H3 mK9 and DNA methylation. Interestingly, suvh5 and suvh6 have different effects at different heterochromatic loci: suvh4 suvh5 causes stronger demethylation of transposons than suvh4 suvh6, but suvh4 suvh6 causes stronger demethylation of a transcribed inverted repeat source of dsRNA than suvh4 suvh5. This locus-specific variation suggests different mechanisms for recruiting or activating SUVH enzymes at different heterochromatic sequences. A triple suvh4 suvh5 suvh6 mutant displays a loss of non-CG methylation similar to a cmt3 mutant, indicating that SUVH4, SUVH5, and SUVH6 together control the majority of CMT3-mediated DNA methylation. |
