Abstract
DNA methylation is a hallmark epigenetic modification extensively involved in regulation of plant molecular and biological processes, most notably genome organization, suppression of transposable elements, and development and environmental responses. While tremendous progress in recent decades has been made in our understanding of epigenetic pathways in plants, the molecular mechanisms of many major proteins and complexes need further elucidation. My thesis focuses on the major plant de novo DNA methyltransferase, DOMAINS REARRANGED METHYLTRANSFERASE 2, or DRM2. DRM2 displays specific domains that are conserved among plants but distinct from DNA methyltransferases of other kingdoms, with partially undetermined structure, interactions, and molecular mechanisms. In my first project, we characterized the DRM2 protein structure and molecular mechanisms behind its regulation. Recent investigations of other DNA methyltransferases frequently found autoinhibition to be a major regulatory mechanism. In vitro experiments confirmed DRM2 autoinhibition and identified putative amino acids that bind and inhibit the catalytic domain. I generated a mutagenized DRM2 transgene and profiled the recovery of DNA methylation when expressing the transgene in the mutant background drm1drm2. Compared to wild-type DRM2, the mutated DRM2 transgene were partially effective at recovering DRM2-dependent methylated regions, while displaying ectopic DNA methylation gains at non-DRM2 methylated targets in the heterochromatin. We hypothesize that introducing these mutations disrupted DRM2 normal recruitment and autoinhibition. In my second project, I discovered a novel interaction between DRM2 with polyubiquitin chain substrates. DRM2 contains three tandemly spaced ubiquitin-associated (UBA) domains that non-covalently bind ubiquitin and polyubiquitin chains. My in vitro experiments with purified DRM2 protein and the tetraubiquitin substrates found that DRM2 DNA methylation activity was increased by addition of polyubiquitin of the K63 type linkage, but not by K48 type linkage. In planta experiments confirmed interaction of DRM2 with K63Ub-containing bodies in the nucleus, and modulations of total K63 ubiquitin homeostasis in plant via its E2 ligase proteins corresponded with changes in DNA methylation of DRM2-dependent targets. Finally, I also performed proximity labeling followed by mass spectrometry to identify potential DRM2 interacting proteins and confirmed several hits by interaction assays in plants. The DRM2-interacting proteins modified by K63Ub will be of great interest for future studies of plant DNA methylation and epigenetic regulation at large.
Committee Chair
Xuehua Zhong
Committee Members
Barbara Kunkel; Jikui Song; Kevin Cox; R.Keith Slotkin; Ram Dixit
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Plant & Microbial Biosciences)
Document Type
Dissertation
Date of Award
8-13-2026
Language
English (en)
DOI
https://doi.org/10.7936/58nc-cp90
Recommended Citation
Le, Huy, "Molecular mechanisms and factors regulating plant DNA methylation" (2026). Arts & Sciences Graduate Student Theses and Dissertations. 3864.
The definitive version is available at https://doi.org/10.7936/58nc-cp90