UC Riverside

Although transposons constitute large portions of eukaryotic genomes, certain mechanisms have evolved to suppress the detrimental effects caused by the movement of transposons. In Arabidopsis, DNA methylation plays a vital role in suppressing transposon expression at the transcriptional level, and the underlying mechanisms have been thoroughly investigated. Numerous factors participating in the DNA methylation pathway have been reported, from the establishment of DNA methylation through RNA-directed DNA methylation (RdDM) to the maintenance of symmetrical DNA methylation by MET1/CMT3 and asymmetrical DNA methylation by RdDM and CMT2.

Despite this well-established framework, however, two important questions remain. The first concerns the mystery precursors to siRNAs that function as guidance signals for RdDM. Although it has been proposed that Pol IV transcribes methylated DNA to produce primary transcripts at RdDM loci and that RDR2 converts these transcripts to dsRNAs to serve as siRNA precursors, no such siRNA precursor transcripts have been reported. In my Ph.D. studies, I was able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci through genome-wide profiling of RNAs in genotypes with compromised siRNA precursor processing. On the one hand, Pol IV/RDR2-dependent transcripts differ from Pol II-dependent transcripts in the following ways: they correspond to both DNA strands instead of one strand, they have a 5' monophosphate instead of a 5' cap, they lack a polyA tail at the 3' end, and they do not have introns. On the other hand, both Pol IV/RDR2-transcribed regions and Pol II-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes. Computational analysis of siRNA abundance in various mutants also revealed differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation through two different DNA methyltransferases.

The second question is how the silencing effect of DNA methylation is controlled to prevent the stochastic silencing of genes or to allow the expression of genes that reside nearby transposons. In my Ph.D. studies, I identified SUVH1 as an anti-silencing factor through a forward genetic screen and showed that it promotes the expression of two transgenes and several endogenous genes. 5-Aza-2'-deoxycitidine (a DNA methylation inhibitor) treatment and methylation level analysis using McrBC-PCR and MethylC-seq subsequently showed that SUVH1 functions downstream of DNA methylation to promote the expression of genes harboring promoter DNA methylation. In addition, SUVH1 was found to maintain H3K4me3 levels. These findings from the functional studies of SUVH1 shed light on the regulatory network acting at genes with various epigenetic marks.