UC Riverside

Cytosine methylation is a key epigenetic mark that mediates diverse biological phenomena by regulating chromatin states. In plants, it is predominantly found in transposons and repeats, and contributes to genome defense through transcriptional gene silencing (TGS). In my thesis research, two genes and their molecular mechanisms in DNA methylation and TGS are uncovered in the model plant Arabidopsis thaliana. First, the LUCH line containing a luciferase (LUC) transgene is characterized as a suitable system for forward genetic screens that aim to isolate genes in TGS. RNA-directed DNA methylation (RdDM), a mechanism that establishes DNA methylation, and active demethylation influence LUC expression by affecting its DNA methylation levels. The moderate LUC expression in LUCH enables genetic screens to isolate both enhancers and suppressors of TGS. Second, TATA-binding proteins-associated factor6 (TAF6) is identified as a positive factor of RdDM and TGS. TAF6 is required for the TGS of the LUCH reporter and several endogenous RdDM targets. Genome-wide methylation assays have uncovered TAF6-dependent loci for proper DNA methylation. TAF6 is well known as a transcription factor of RNA polymerase II (Pol II), however, I find that a TAF6 mutation compromises the transcription by a Pol II homolog Pol V. These findings indicate that Pol V adopts the Pol II regulatory mechanism to generate long non-coding RNAs that guide RdDM. Third, a heat shock protein 20 (HSP20) is uncovered as a negative regulator of DNA methylation and TGS. HSP20 prevents the silencing of TGS reporters as well as of several endogenously methylated targets. Mutations in HSP20 result in hypermethylation and mislocalization of the RdDM effector protein ARGONAUTE4 in the nucleus. Moreover, HSP20 is found to interact with three methyl CpG-binding domain (MBD) proteins that may link cytosine methylation to histone modifications. Mutation in MBD5 or MBD6 also hypermethylated several HSP20-dependent loci. Based on these findings, I propose that HSP20 acts with MBDs to recognize loci that undergo DNA methylation and antagonize TGS at these loci. While HSP20-MBDs likely act downstream of DNA methylation, they also affect the DNA methylation status through direct or indirect effects, such as AGO4 recruitment or histone modifications.