Studies On Methylation Reactions In Plants

Author : Ping-Hung Hsieh
Publisher :
ISBN 13 :
Total Pages : 84 pages
Book Rating : 4.:/5 (994 download)

DOWNLOAD NOW!

Download or read book Maintenance and Inheritance of DNA Methylation in Arabidopsis written by Ping-Hung Hsieh and published by . This book was released on 2016 with total page 84 pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA methylation is a conserved epigenetic modification, usually of the 5th carbon of cytosine in eukaryotes, and in plants is known to regulate gene expression and silence transposable elements. Posttranslational histone tail modifications are also conserved epigenetic regulators, known in the plant kingdom to interact with DNA methylation and regulate chromatin structure. Both DNA methylation and histone modifications are reversible and, collectively, play an important role in the orchestration of dynamic transcriptional profiles during the entire life cycle of the plant. In plants, DNA methylation is found in the symmetric CG and CHG contexts, and the asymmetric CHH context (where H is A, T or C). Several studies have shown that CG methylation is catalyzed by the mammalian DNMT1 methyltransferase ortholog, MET1, and CHG methylation is maintained by the plant specific chromomethylase 3 (CMT3), through a self-reinforcing loop between CMT3 and the heterochromatic dimethylation of lysine 9 of the histone H3 subunit (H3K9me2). Due to its asymmetry, CHH methylation was thought to be maintained solely by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2, an ortholog of mammalian DNMT3-type methyltransferases) through the plant-specific RNA-directed DNA methylation (RdDM) pathway. However, DRM2-mediated CHH methylation only accounts for about 35% of total CHH methylation in Arabidopsis, suggesting that other proteins contribute to the maintenance of CHH methylation. To identify which proteins are involved in the maintenance of CHH methylation in addition to DRM2, I performed a reverse genetic screen on Arabidopsis mutants to identify genes that can potentially affect plant DNA methylation by performing whole-genome bisulfite sequencing of each individual homozygous mutant seedling. Eventually, I found that CMT2, a homolog of CMT3, is an active methyltransferase that maintains ~70% of total CHH methylation in Arabidopsis, independently of RdDM. Furthermore, I determined that the DRM2-mediated RdDM pathway mainly carries out CHH methylation in more euchromatic regions, including short TEs in chromosome arms and the edges of long TEs in pericentromeric regions, whereas CHH methylation at more heterochromatic sites, such as the bodies of long TEs, is mediated by CMT2. This maintenance activity is thought to allow DNA methylation to carry epigenetic information through cell division and reproduction, influencing gene expression and phenotype across generations. Trans-generational inheritance is mediated by a small group of cells that includes gametes and their progenitors in flowering plants. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of trans-generational inheritance are inferred from such studies. The male gametophyte pollen, consisting of two sperm cells and a vegetative cell, was reported to undergo DNA reprogramming during sexual reproduction: a gain of heterochromatic CHH methylation in vegetative cells and a loss of heterochromatic CHH methylation in sperm cells were attributed to the activation of DRM2 and the silencing of DRM2, respectively. To gain further insight into how DNA methylation is inherited and reprogrammed during sexual reproduction, I analyzed Arabidopsis thaliana sperm and vegetative cells purified from pollen with mutations in the DRM (both DRM1 and DRM2. DRM1 is specifically expressed during early seed development and functions redundantly with DRM2), CMT2, and CMT3 methyltransferases. I found that although the gain of heterochromatic CHH methylation in the vegetative cell is primarily mediated by CMT2 instead of DRM, the effect of the cmt2 mutation in the vegetative cell was weaker than expected, and a small but significant fraction of new CHH methylation targets in heterochromatic TEs was mediated by DRM through RdDM pathway, which supports the observation that many heterochromatic TEs are derepressed in the vegetative cell. I also showed that lack of histone H1, which elevates heterochromatic DNA methylation in somatic tissues, does not have this effect in pollen. Instead, levels of CG methylation in wild-type sperm and vegetative cells, as well as in wild-type microspores from which both pollen cell types originate, are substantially higher than in wild-type somatic tissues and are similar to those of H1-depleted roots. In summary, I discovered a novel DNA methyltransferase in plants, CMT2, which maintains CHH methylation independently from RdDM. I showed that the DNA methylation of germlines are maintained similarly to somatic tissues, which further explains why the reprogramming of CHH methylation in pollen is most likely dependent on CMT2. Although the mechanisms of methylation maintenance are similar between pollen and somatic cells, I demonstrated that the efficiency of CG methylation is higher in pollen, allowing methylation patterns to be accurately inherited across generations. The biological significance of the reduced CHH methylation found in sperm remains unknown. Overall, my findings expand the previous incomplete DNA methylation maintenance model with the discovery of CMT2-mediated CHH methylation. My work on the pollen methylome contributes to our further understanding of the regulation and reprogramming of DNA methylation during sexual reproduction, providing insights into transgenerational epigenetic inheritance in plants.