DNA methylation is one of the most well studied repressive epigenetic marks in eukaryotes. In plants, DNA methylation silences genes and transposons, and establishes genomic imprinting. Genomic imprinting is the mono-allelic expression of a gene occurring in a parent-of-origin specific manner. Imprinted genes tend to be expressed in nutritive tissues and structures that function to support the growing embryo. In mammals, this is the placenta, and in plants this is the endosperm.
In Arabidopsis, the female gametophyte contains the egg and central cell, while the male gametophyte contains the vegetative cell and two sperm cells. Both the central cell and vegetative cells are companion cells to the egg and sperm cells respectively. When pollen lands on specialized cells at the tip of the gynoecium, the vegetative cell forms a pollen tube that transports the two sperm cells to the ovule. Within the ovule, one sperm fertilizes the egg cell to form the embryo, whereas a second sperm fertilizes the central cell to form the endosperm, which supports embryo development by providing nutrients.
In Arabidopsis, the DME DNA glycosylase carries out active DNA demethylation in the central cell of the female gametophyte, which has been shown to establish genomic imprinting in Arabidopsis. Recently, DME-mediated DNA demethylation has also been shown to occur in the vegetative cell of the male gametophyte, as well.
The aim of the work carried out in my thesis has been to investigate the role of DNA methylation and DME-mediated DNA demethylation in the central cell and vegetative cell. In particular, I was interested in identifying and understanding the regions in the genome where DME-mediated DNA demethylation occurs. My overall approach was to perform high-throughput bisulfite sequencing using the illuminaTM platform on the Arabidopsis endosperm, embryo, and dme-mutant endosperm, and to analyze their DNA methylation profiles (methylomes). In my initial study (Chapter II), I measured non-allele-specific methylomes in which endosperm and embryo dissected from selfed-plants were used. In my subsequent study (Chapter III), I determined allele-specific (maternal versus paternal) methylomes by analyzing endosperm and embryo derived from seeds generated by crossing different Arabidopsis ecotypes. Because there are no current methods for determining the Arabidopsis central cell methylome, I sought to use the maternal endosperm genome as a proxy for the maternal central cell genome. Through collaboration, I also had the opportunity to investigate and explore the methylomes of wild-type and dme-mutant vegetative cell and sperm cells, which enabled me to compare the maternal and paternal reproductive methylomes of the plant.
To the best of my knowledge, this is the first time a high-throughput DNA methylation study was done on the endosperm of a flowering plant. With my collaborators, we discovered that the Arabidopsis endosperm exhibits a genome-wide reduction in DNA methylation compared to the embryo. We also found that the DME DNA glycosylase regulates non-CG methylation in the endosperm, particularly siRNA-mediated CHH methylation. Moreover, our analysis of DME-dependent DNA demethylation in companion cells suggested a mechanism for reinforcing silencing of transposons in the plant male and female gamete cells.
We found that DME demethylated many of the same loci in both the maternal endosperm and vegetative cell genomes, suggesting that DME has a much more robust and primitive role in Arabidopsis reproduction than previously thought. Interestingly, many of these DME-dependent sites were heavily enriched in small transposons and repetitive elements. Moreover, these small transposons tended to reside adjacent to genes, enabling us to propose a new model for the observed phenomenon of gene-adjacent DNA demethylation in Arabidopsis. That is, regulation of gene imprinting may not be the basal function of DME demethylation. These studies suggest that the primary function of demethylation of transposons in companion cells may be to reinforce transposon silencing in plant gametes.
